Conjugates of membrane translocating agents and pharmaceutically active agents

ABSTRACT

Membrane translocation peptides, compositions comprising them, chimeric molecules comprising them, and methods of using them to achieve transmembrane transport of various agents.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application Ser.No. 09/671,089 filed Sep. 27, 2000, incorporated by reference herein inits entirety, and also claims the benefit of U.S. provisionalapplication serial No. ______ filed Apr. 30, 2001 with the title“Lipid-Comprising Drug Delivery Complexes and Method for TheirProduction”, incorporated herein in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to peptides, which enhance uptakeof a pharmaceutically active agent into a cell, into or out of anintracellular compartment, and across a cell layer. More particularly,the present invention relates to membrane translocating peptides,thereof and to the nucleotide sequences coding therefor, which enhanceuptake of a pharmaceutically active agent into a cell, into or out of anintracellular compartment, and across a cell layer either directly orfrom a pharmaceutically active agent loaded particle.

BACKGROUND OF THE INVENTION

[0003] The epithelium lining the gastrointestinal tract (hereinafter,“GIT”) is a major barrier to absorption of orally administeredpharmaceutically active agents (hereinafter, “active agents”).Absorption across the GIT epithelium can be by transcellular transportthrough the cells and by paracellular transport between the cells.Transcellular transport includes, but is not limited to,receptor-mediated, transporter-mediated, channel-mediated, pinocytoticand endocytotic mechanisms and to diffusion. Paracellular transportincludes, but is not limited to, movement through tight junctions. Ofparticular interest is the development of non-invasive methods forenhancing uptake of active agents across the GIT epithelium into thebody (Evers, P. Developments in Drug Delivery: Technology and Markets,Financial Times Management Report, 1995).

[0004] To develop non-invasive methods, phage display libraries havebeen used to identify specific peptide sequences, which bindpreferentially to specific GIT membrane receptor, transporter, channel,pinocytotic or endocytotic target pathways (hereinafter, “targetingpeptides”) within the GIT. Included among the target pathways, whichhave been screened with phage display libraries, are the GIT membranetransporters HPT1, hPEPT1, D2H and hSI. HPT1 and hPEPT1 transportdipeptides and tripeptides. D2H transports neutral and basic amino acidsand is a transport activating protein for a range of amino acidtranslocases. hSI is involved in sugar metabolism and comprises 9% ofthe brush border protein in the jejunum. Specific peptide sequences,which interact with the HPT1, hPEPT1, D2H and hSI membrane transportershave been identified in the following 4 applications, each of which isincorporated herein in its entirety: U.S. patent applications Ser. Nos.09/079,819, 09/079,723 and 09/079,678, and PCT application,PCT/US98/10088, published as WO 98/51325.

[0005] Non-target pathway based assays have been used to identifypeptides with inherent cell membrane translocating properties. Thesecell membrane translocating peptides interact directly with andpenetrate the lipids of cell membranes (Fong et al. Drug DevelopmentResearch 33:64, 1994). The central hydrophobic h-region of the signalsequence of Kaposi's fibroblast growth factor, AAVLLPVLLAAP (SEQ IDNO: 1) is considered to be a membrane translocating peptide. Thispeptide (SEQ ID NO: 1) has been used as a carrier to deliver variousshort peptides (<25 mer), through the lipid bilayer, into living cellsin order to study intracellular protein functions and intracellularprocesses (Lin et al. J. Biol. Chem. 271:5305,1996; Liu et al. Proc.Natl. Acad. Sci. USA 93:11819, 1996; Rojas et al. J. Biol. Chem.271:27456, 1996; Rojas et al. Biochem. Biophys. Res. Commun.234:675,1997). A 41-kDa glutathione S-transferase fusion proteincontaining SEQ ID NO: 1 (GST-Grbs-SH₂fused to SEQ ID NO: 1) has beenshown to be imported into NIH 3T3 fibroblasts and to inhibit epidermalgrowth factor induced EGFR-Grb2 association and MAP kinase activation(Rojas et al. Nature Biotechnology 16:370, 1998). However, these studiesdo not address the use of membrane translocating peptides to enhanceactive agent uptake into a cell, into and out of an intracellularcompartment, or across a cell layer when the active agent is complexedto a membrane translocating peptide or when the active agent isincorporated into a particle and the particle is modified with(hereinafter, “complexed to”) a membrane translocating peptide.

[0006] The ability to enhance movement of an active agent across a cellmembrane is important because, although an active agent can beadministered to an animal by a variety of routes including, but notlimited to, oral, nasal, mucosal, topical transdermal, intravenous,intramuscular, intraperitoneal, intrathecal and subcutaneous, oraladministration is the preferred route. Nasal, mucosal, topical andtransdermal administration depend on drug absorption through the mucosaor skin into the circulation. Intravenous administration can result inadverse effects from rapid accumulation of high concentrations of drug,in patient discomfort and in infection at the injection site.Intramuscular administration can cause pain at the injection site.Subcutaneous administration is not suitable for large volumes or forirritating substances. Although oral administration is the preferredroute, many active agents are not absorbed efficiently across the GITepithelium. This results from enzymatic degradation of active agentswithin the lumen of the GIT, from the limited permeability of the GITepithelium to active agents, from the large molecular size of activeagents and from the hydrophilic properties of active agents (Fix, JA. J.Pharmac. Sci. 85:1282, 1996). To develop an oral formulation, an activeagent must be protected from enzymatic digestion within the lumen of theGIT, presented to the absorptive epithelial cells of the GIT in aneffective concentration and “moved” across the epithelium in an apicalto basolateral direction.

[0007] Therefore, because of the advantages of oral drug administration,there is a need for delivery systems, which protect orally ingestedactive agents from enzymatic degradation within the lumen of the GIT andwhich promote the absorption of orally ingested active agents into andacross the epithelial cells lining the GIT.

BRIEF SUMMARY OF THE INVENTION

[0008] The present invention fulfills the above-noted needs by providingmembrane translocating peptides (hereinafter referred to interchangeablyeither as “MTLPs” or “translocating peptides”) or nucleotide sequencescoding therefore, MTLP-active agent complexes and MTLP-active particlecomplexes, wherein the MTLP enhances movement of the active agent or theactive particle across a lipid membrane. More particularly, the presentinvention provides a MTLP, MTLP-active agent complexes and MLTP-activeparticle complexes, wherein the MTLP enhances movement of the activeagent or of the active particle into a cell, into and out of anintracellular compartment and across a cell layer in an animal,including a human. Methods of making and methods of using MTLPs,MTLP-active agent complexes and MTLP-active particle complexes also areincluded.

[0009] Compositions and Their Peptides

[0010] More precisely, in a first general aspect, the invention is acomposition comprising a translocating peptide, said translocatingpeptide selected from the group consisting of a transport peptide, anextended peptide comprising said transport peptide, and atransport-active fragment of at least 4 amino acids of said transportpeptide, wherein said transport peptide is selected from the groupconsisting of an L-peptide, a d-peptide, and a retroinverted peptide,and

[0011] wherein said L-peptide has an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 2-13, and 15-24,

[0012] wherein said d-peptide has an amino acid sequence selected fromthe group consisting of SEQ ID NOS. 102-124 corresponding to the d-formsof L-peptides of SEQ ID NOS. 2-24, and

[0013] wherein the retroinverted peptide has an amino acid sequenceselected from the group consisting of a peptide of SEQ ID NOS. 202-224,corresponding to retroinverted forms of L-peptides of SEQ ID NOS: 2-24.

[0014] A specific embodiment of the composition (first general aspect)is one wherein terminal or near-terminal lysines play a role:

[0015] the L-peptide has an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 2-4, 16, 23 and 24.

[0016] the d-peptide has an amino acid sequence selected from the groupconsisting of SEQ ID NOS. 102-104, 116, 123 and 124 corresponding to thed-forms of L-peptides of SEQ ID NOS. 2-4, 16, 23 and 24, and

[0017] the retroinverted peptide has an amino acid sequence selectedfrom the group consisting of a peptide of SEQ ID NOS. 202-204, 216, 223and 224 corresponding to retroinverted forms of an L-peptides of SEQ IDNOS: 2-4, 16, 23 and 24.

[0018] In another specific embodiment of the composition, the transportpeptide is partially or completely cyclic. In a related embodiment, anyfragment of the transport peptide is also partially or completelycyclic. Cyclic peptides of particular interest are those in which

[0019] the L-peptide has an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 5-13;

[0020] the d-peptide has an amino acid sequence selected from the groupconsisting of SEQ ID NOS. 105-113 corresponding to the d-forms ofL-peptides of SEQ ID NOS. 5-13, and

[0021] the retroinverted peptide has an amino acid sequence selectedfrom the group consisting of a peptide of SEQ ID NOS. 205-213,corresponding to retroinverted forms of L-peptides of SEQ ID NOS: 5-13.

[0022] In another particular embodiment of the composition, thetranslocating peptide is an extended peptide of a transport peptide.Preferably, the extended peptide is not more than 100 amino acids inlength, more preferably not more than 50 amino acids in length.

[0023] In a further embodiment of the composition, the translocatingpeptide is a transport peptide.

[0024] It is preferred that the transport-active fragment is at least 6amino acids, more preferably 8 amino acids of a transport peptide.

[0025] In preferred embodiments, the carboxyl end group of thetranslocating peptide is one that has been modified to create an amidegroup.

[0026] Closely related to the above compositoins is one comprising atranslocating peptide, said translocating peptide selected from thegroup consisting of a transport peptide, an extended peptide comprisingsaid transport peptide, and a transport-active fragment of at least 4amino acids of said transport peptide, said transport peptide being anL-peptide that has an amino acid sequence SEQ ID NO: 14 blocked at itscarboxyl end with an amide group and wherein any of said fragments isalso blocked at its carboxyl end with an amide group.

[0027] The foregoing compositions can, for example, further comprise anactive agent, wherein the translocating peptide is complexed to anactive agent to form a translocating peptide-active agent complex.

[0028] Additionally, the compositions can, for example, further comprisean active particle, wherein the translocating peptide is complexed tothe active particle to form a translocating peptide-active particlecomplex.

[0029] Chimeric Peptides

[0030] Chimeric polypeptides comprising the translocating peptides arealso part of the invention. Specifically such polypeptides comprise (A)a translocating peptide of this invention, (B) a translocatable peptide,and (C) an amino acid linker sequence that directly linkd thetranslocating peptide to the translocatable peptide, wherein saidtranslocatable peptide is between 3 and 200 amino acids, and whereinsaid amino acid linker sequence is between 1 and 20 amino acids.

[0031] In particular embodiments of the chimeric peptides, thetranslocatable peptide is between 3 and 30 amino acids.

[0032] In other embodiments, the translocatable peptide is an opioidpeptide (examples of which are listed elsewhere herein).

[0033] In some particular embodiments, the linker sequence is not morethan 7 amino acids, preferably not more than 3 amino acids. In someuseful embodiments, the linker sequence is 1 amino acid.

[0034] In some preferred embodiments least 50% of the amino acids in thelinker sequence are lysines. More preferably at least 80% of the aminoacids in the linker sequence are lysines. Most preferably all of theamino acids in the linker sequence are lysines.

[0035] Chimeric Constructs

[0036] Closely related to the chimeric peptides of this invention arechimeric constructs. Such constructs comprise (A) a translocatingpeptide of this invention, (B) a translocatable peptide, and (C) anon-amino acid linker that directly links the translocating peptide tothe translocatable peptide, wherein said translocatable peptide isbetween 3 and 200 amino acids.

[0037] Preferred non-amino acid linkers are those that have a molecularweight of less than 1000 (more preferably less than 500).

[0038] Preferred non-amino acid linkers are those that provide thechimeric construct at least 50% (more preferably at least 100%) of thestability in SIF as a single lysine linker does for the correspondingchimeric peptide when the translocating peptide is Elan207 and thetranslocatable peptide is the kappa opioid peptide (at 37° C. for 1hour, where stability is indicated by retention of the structure of thechimeric structure or peptide.)

[0039] Preferred non-amino acid linkers are those that provide thechimeric construct with an IC50 that is not more than twice that of thecorresponding chimeric peptide when the translocating peptide is Elan207and the translocatable peptide is the kappa opioid peptide and the IC50is measured in the radio-labelled kappa peptide rat brain homogenateassay described herein.

[0040] Two or more of the above preferred aspects for a non-amino acidlinker is even more preferable.

[0041] Examples of non-amino acid linkers are:

[0042] Hydrocarbon chains which can include both unsubstituted andsubstituted alkyl, aryl, or macrocyclic R groups. Alkyl is intended tomean any straight, branched, saturated, unsaturated or cyclic C1-20alkyl group. Aryl is intended to mean any aromatic cyclic hydrocarbonbased on a six-membered ring. Macrocycle refers to R groups containingat least one ring containing more than seven carbon atoms. Substitutedis intended to mean any alkyl, aryl, or macrocyclic groups in which atleast one carbon atom is covalently bonded to any functional groupscomprising the atoms H. C, N, O, S, F, Cl, Br and I. For details seeApplication PCT/US00/23440 published as WO 01/01/13957, pages 5-7 ofwhich are incorporated by reference herein in their entirety).

[0043] Additional possible linkers are summarized in PCTapplication/US99/13660 published as WO 99/67284, especially pages 21-23;

[0044] Some linkers are well-suited for situations where cleavage of thelinker is desired only at specific sites in a person, for example,specific tissue, specific fluid, specific cells, or specificsub-cellular compartments. Examples of where some of the linkers displaysuch cleavage specificity is denoted in parentheses after those linkers,as follows:

[0045] Amide (amidase sensitive)

[0046] Carbamate (stable in plasma, triggered release)

[0047] Disulphide (stable in plasma, reduced in cell compartments,reduced during crossing of BBB)

[0048] Ester (pH sensitive, esterase sensitive)

[0049] Carbonate (pH sensitive, non-specific enzymatic degradation)

[0050] Methods of the Invention

[0051] Related to the compositions of the invention are methods thatutilize them.

[0052] One method of the invention is one that delivers a chimericpeptide to the blood, said method comprising orally administering achimeric peptide of the invention.

[0053] Another method of the invention is a method of delivering achimeric construct to a site within a person, said method comprisingadministering a chimeric construct of this invention, said site beingselected from the group consisting of a tissue, a fluid, a cell, and asub-cellular compartment.

[0054] Another method of the invention is for enhancing movement of anactive agent across a lipid membrane, which comprises using atranslocating peptide-active agent complex, wherein the translocatingpeptide enhances movement of the active agent across the lipid membrane.

[0055] Another method of the invention is one for enhancing movement ofan active particle across a lipid membrane, which comprises using atranslocating peptide active particle complex, wherein the translocatingpeptide enhances movement of the active particle across the lipidmembrane.

[0056] Still another method of the invention is one for identifying acompound having enhanced ability to transport an active agent across alipid membrane, wherein the compound competes with the translocatingpeptide for transport across a membrane selected from the groupconsisting of a cell membrane, an intracellular membrane, the apical andbasal membranes of an epithelial cell layer. In a particular embodiment,the epithelial cell layer is a polarized epithelial cell layer.

[0057] Another method of the invention is one for treating apathological disorder in an animal, comprising orally administering tothe animal in need of such treatment a complex selected from the groupconsisting of a translocating peptide-active agent complex and atranslocating peptide-active particle complex, wherein an amount of theactive agent effective to treat the pathological disorder is movedacross the gastrointestinal epithelium of the animal into thecirculation.

[0058] MTLPs of the present invention are capable of displaying one ormore known functional activities associated with a full-length MTLP.Such functional activities include, but are not limited to, the abilityto interact with a membrane and the ability to compete for transport ofa reporter drug molecule (fMLP) across epithelial cells including, butnot limited to, polarized, differentiated human derived Caco-2 cells.Additional functional activities include, but are not limited to,antigenicity, which includes, but is not limited to, the ability to bindto an anti-MTLP antibody and the ability to compete with a MTLP forinteraction with a membrane; and, immunogenicity, which includes, but isnot limited to, the ability to stimulate antibody generation.

[0059] Methods of making a MTLP-active agent complex include, but arenot limited to, covalent coupling of a MTLP and an active agent andnoncovalent coupling of a MTLP and an active agent. Methods of making aMTLP-active particle complex include, but are not limited to,incorporating an active agent into a particle including, but not limitedto, a nanoparticle, a microparticle, a capsule, a liposome, a non-viralvector system and a viral vector system. The MTLP can be complexed tothe active particle by methods including, but not limited to, adsorptionto the active particle, noncovalent coupling to the active particle andcovalent coupling, either directly or via a linker, to the activeparticle, to the polymer or polymers used to synthesize the activeparticle, to the monomer or monomers used to synthesize the polymer, andto other components comprising the active particle.

[0060] The present invention also includes the nucleotide sequences,which code for the MTLPs. Methods of making nucleotide sequencesinclude, but are not limited to, recombinant means.

[0061] MTLPs, MTLP-active agent complexes and MTLP-active particlecomplexes can be used alone, in combination with or conjugated to othermolecules including, but not limited to, molecules that bind to targetpathways, to nuclear uptake pathways and to endosomal pathways,molecules that enable mucoadhesion, molecules that facilitate diffusionacross lipid membranes or through water filled pores and molecules thatregulate or direct intra-cellular trafficking. That is, by usingdifferent mechanisms simultaneously, active agent bioavailability may beenhanced.

[0062] Related inventions are the use of translocating peptides (i.e.,MTLPs) in the following:

[0063] a composition comprising a translocating peptide-active particlecomplex, wherein the particle is a microparticle;

[0064] a composition comprising a translocating peptide-active particlecomplex, wherein the particle is a nanoparticle;

[0065] a composition comprising a translocating peptide-active particlecomplex, wherein the particle is a liposome;

[0066] a composition comprising a viral DNA particle, wherein the viralparticle is modified to express a translocating peptide on its surface;

[0067] a composition comprising a viral DNA particle, wherein the viralparticle is complexed to a translocating peptide following virusproduction and purification;

[0068] a composition comprising a viral DNA particle, wherein the viralparticle is complexed to a translocating peptide following virusproduction in and purification from a mammalian cell; and

[0069] a composition comprising a non-viral based gene delivery system,wherein the non-viral based gene delivery system is complexed to atranslocating peptide.

[0070] Further related inventions are the use of translocating peptidesin the following methods:

[0071] a method to enhance the movement of an active agent across alipid membrane;

[0072] a method to enhance the uptake of an active agent into a cell;

[0073] a method to enhance the uptake of an active agent across a celllayer;

[0074] a method to enhance the uptake of an active agent into anepithelial cell;

[0075] a method to enhance the uptake of an active agent across anepithelial cell layer;

[0076] a method to enhance the uptake of an active agent across theepithelial cell layer lining the GIT into the circulation of an animal;

[0077] a method to enhance the movement of an active particle across alipid membrane;

[0078] a method to enhance the uptake of an active particle into a cell;

[0079] a method to enhance the uptake of an active particle across acell layer;

[0080] a method to enhance the uptake of an active particle into anepithelial cell;

[0081] a method to enhance the uptake of an active particle across anepithelial cell layer;

[0082] a method to enhance the uptake of an active particle across theepithelial cell layer lining the GIT into the circulation of an animal;

[0083] a method to provide intracellular gene delivery by a non-viralbased gene delivery system;

[0084] a method to provide intracellular gene delivery by a non-viralbased gene delivery system, wherein the non-viral based gene deliverysystem is complexed to a translocating peptide;

[0085] a method to provide a rapid screening method to identifytranslocating peptides, which retain the essential functional activityof the full-length translocating peptide;

[0086] a method to provide cell-based screens for assaying thefunctional activity of; and

[0087] a method to provide cell-based screens for characterizing theproperties of a translocating peptide.

[0088] Another aspect of the present invention is a method to provide amethod for diagnosing a pathological disorder by oral administration ofan amount of a translocating peptide-active agent complex, wherein theactive agent is a diagnostic agent, such that the systemic concentrationof the diagnostic agent is effective to diagnose the pathologicaldisorder.

[0089] Another aspect of the present invention is a method to provide amethod for preventing a pathological disorder by oral administration ofa translocating peptide-active agent complex, wherein the active agentis a prophylactic agent, such that the systemic concentration of theprophylactic agent is effective to prevent the pathological disorder.

[0090] Another aspect of the present invention is a method for treatinga pathological disorder by oral administration of a translocatingpeptide-active agent complex, wherein the active agent is a therapeuticagent, such that the systemic concentration of the therapeutic agent iseffective to treat the pathological disorder.

[0091] Another aspect of the present invention is a method to provide amethod for diagnosing a pathological disorder by oral administration ofa translocating-active particle complex, wherein the active particlecontains a diagnostic agent, such that the systemic concentration of thediagnostic agent is effective to diagnose the pathological disorder.

[0092] Another aspect of the present invention is a method to provide amethod for preventing a pathological disorder by oral administration ofa a translocating peptide-active particle complex, wherein the activeparticle contains a prophylactic agent, such that the systemicconcentration of the prophylactic agent is effective to prevent thepathological disorder.

[0093] Another aspect of the present invention is a method to provide amethod for treating a pathological disorder by oral administration of aa translocating peptide-active particle complex, wherein the activeparticle contains a therapeutic agent such that the systemicconcentration of the therapeutic agent is effective to treat thepathological disorder.

[0094] Other objectives, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0095]FIG. 1 shows the hydropathy plot for ZElan094 (16 mer) (SEQ ID NO:2);

[0096]FIG. 2 shows the systemic blood insulin levels following in vivodelivery of insulin from a ZElan094-insulin nanoparticle complex andfrom HAX42-, PAX2- and P31-insulin nanoparticle complexes in the openloop rat model. Each point is the mean of 6-7 animals;

[0097]FIG. 3 shows the systemic blood glucose levels following in vivodelivery of insulin from a ZElan094-insulin nanoparticle complex andfrom HAX42-, PAX2- and P31-insulin nanoparticle complexes in the openloop rat model. Each point is the mean of 6-7 animals;

[0098]FIG. 4 shows the transport of the reporter drug ³H-fMLP acrossCaco-2 monolayers in the presence of the MTLPs ZElan094, 178, 187 andthe targeting peptide ZElan022;

[0099]FIG. 5 shows the transport of the reporter drug ³H-fMLP acrossCaco-2 monolayers in the presence of increasing concentrations of theMTLP ZElan094.

[0100]FIG. 6 shows the transport of ³H-Kappa peptide conjugates acrossCaco-2 monolayers.

DETAILED DESCRIPTION OF THE INVENTION

[0101] The present invention relates to novel membrane translocatingpeptides (MTLPs or, alternatively, “translocating peptides”) tonucleotide sequences coding therefor, to MTLP-active agent complexes andto MTLP-active particle complexes, wherein the MTLP enhances movement ofthe active agent or of the active particle across a membrane. Moreparticularly, the present invention relates to novel MTLPs, tonucleotide sequences coding therefore, to MTLP-active agent complexesand to MTLP-active particle complexes, wherein the MTLP enhancesmovement of the active agent in the MTLP-active agent complex, of theactive agent in the MTLP-active particle complex and of the activeparticle in the MTLP active-particle complex into a cell, into and outof an intracellular compartment and across a cell layer in an animal,including a human. Methods of making and methods of using MTLPs also areincluded.

[0102] The present invention also provides methods for diagnosing,preventing or treating a pathological disorder in an animal in need ofdiagnosis, prevention or treatment of a pathological disorder byadministrating to the animal an amount of a MTLP-active agent complex orof a MTLP-active particle complex, such that the systemic concentrationof the active agent is effective to diagnose, prevent or treat thepathological disorder.

[0103] An “active agent”, as used herein, includes any diagnostic,prophylactic or therapeutic agent that can be used in an animal,including a human.

[0104] An “active particle”, as used herein is a particle into which oneor more active agents have been loaded.

[0105] A membrane translocating peptide, as used herein, is a peptidewhich interacts directly with and penetrates the lipids of aphysiological membrane.

[0106] A “MTLP”, as used herein, is a general term that refers to anytranslocating peptides refered to herein. Specific MTLPs where sequencesare described herein can be part of larger peptides or polypeptides, allwhich, in turn, are MTLPs. Transport-active fragements of MTLPs are alsoMTLPs.

[0107] A “transport-active fragment” of a translocating peptide is onethat increases the plasma ³H bioavailability of a Kappa peptide by 30%,compared to the Kappa peptide above after intraduodenal installation inthe Wistar rat model described herein in Example 15.

[0108] The terms “peptide” and “polypeptide” are used to some extentinterchangebly herein and no precise size demarcation between the two isintended.

[0109] A “composition comprising a translocating peptide” could includenot only homogeneous composition consisting only of particular peptide,but also compositions that contain additional components, includingend-group moities that are covalently linked to the amino or carboxylend of such translocating peptides. Specific examples of such inend-group moieties, such as amide, dansyl and biotin groups, areprovided herein.

[0110] The term “translocating peptide” is used for convenience inphrasing claims that refer to a group of various possible transportpeptides. No biochemical difference in function between translocatingand transport peptides is intended.

[0111] “Complexed to”, as used herein, includes adsorption, non-covalentcoupling and covalent coupling of a MTLP to an active agent or to anactive particle.

[0112] A “MTLP-active agent complex”, as used herein, includes one ormore MTLPs complexed to an active agent.

[0113] A “MTLP-active particle complex”, as used herein, includes one ormore MTLPs complexed to an active particle.

[0114] The active agent used depends on the pathological condition to bediagnosed, prevented or treated, the individual to whom it is to beadministered, and the route of administration. Active agents include,but are not limited to, imaging agents, antigens, antibodies,oligonucleotides, antisense oligonucleotides, genes, gene correctinghybrid oligonucleotides, aptameric oligonucleotides, triple-helixforming oligonucleotides, ribozymes, signal transduction pathwayinhibitors, tyrosine kinase inhibitors, DNA-modifying agents,therapeutic genes, systems for therapeutic gene delivery, drugs andother agents including, but not limited to, those listed in the UnitedStates Pharmacopeia and in other known pharmacopeias

[0115] Drugs include, but are not limited to, peptides, proteins,hormones and analgesics, cardiovascular, narcotic, antagonist,chelating, chemotherapeutic, sedative, anti-hypertensive, anti-anginal,anti-migraine, anti-coagulant, anti-emetic anti-neoplastic andanti-diuretic agents Hormones include, but are not limited to, insulin,calcitonin, calcitonin gene regulating protein, atrial natriureticprotein, colony stimulating factor, erythropoietin (EPO), interferons,somatotropin, somatostatin, somatomedin, luteinizing hormone releasinghormone (LHRH), tissue plasminogen activator (TPA), growth hormonereleasing hormone (GHRH), oxytocin, estradiol, growth hormones,leuprolide acetate, factor VIII, testosterone and analogs thereof.Analgesics include, but are not limited to, fentanyl, sufentanil,butorphanol, buprenorphine, levorphanol, morphine, hydromorphone,hydrocodeine, oxymorphone, methadone, lidocaine, bupivacaine,diclofenac, naproxen, paverin, and analogs thereof. Anti-migraine agentsinclude, but are not limited to heparin, hirudin, and analogs thereof.Anti-coagulant agents include, but are not limited to, scopolamine,ondansetron, domperidone, etoclopramide, and analogs thereof.Cardiovascular, anti-hypertensive and vasodilator agents include, butare not limited to, diltiazem, clonidine, nifedipine, verapamil,isosorbide-5-mononitrate, organic nitrates, nitroglycerine and analogsthereof. Sedatives include, but are not limited to, benzodiazeines,phenothiozines and analogs thereof. Narcotic antagonists include, butare not limited to, naltrexone, naloxone and analogs thereof. Chelatingagents include, but are not limited to deferoxamine and analogs thereof.Anti-diuretic agents include, but are not limited to, desmopressin,vasopressin and analogs thereof. Anti-neoplastic agents include, but arenot limited to, 5-fluorouracil, bleomycin, vincristine, procarbazine,temezolamide, CCNU, 6-thioguanine, hydroxyurea and analogs thereof.

[0116] An active agent can be formulated in neutral or salt form.Pharmaceutically acceptable salts include, but are not limited to, thoseformed with free amino groups; those formed with free carboxyl groups;and, those derived from sodium, potassium, ammonium, calcium, ferrichydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidineand procaine. An active agent can be loaded into a particle preparedfrom pharmaceutically acceptable ingredients including, but not limitedto, soluble, insoluble, permeable, impermeable, biodegradable orgastroretentive polymers or liposomes. Such particles include, but arenot limited to, nanoparticles, biodegradable nanoparticles,microparticles, biodegradable microparticles, nanospheres, biodegradablenanospheres, microspheres, biodegradable microspheres, capsules,emulsions, liposomes, micelles and viral vector systems.

[0117] MTLPs have functional activities. Such functional activitiesinclude, but are not limited to, enhancing uptake of an active agentinto a cell, into and out of an intracellular compartment and across acell layer and competing with the full-length peptide in enhancinguptake of an active agent into a cell, across a cell layer or into andout of an intracellular compartment.

[0118] Examples of MTLPs of the present invention include, but are notlimited, to those containing as primary amino acid sequences, all orpart of the amino acid sequences substantially as depicted in Table 1TABLE 1 MTLPs Amino acid sequences SEQUENCE + K(ε-dansyl) ELAN NO. SEQID NO. H-K(ε-dansyl)KKAAAVLLPVLLAAP-NH2 ZElan094  2H-KKAAAVLLPVLLAAP-FITC-LC-NH2 FElan094  3H-K(ε-dansyl)KKAAAVLLPVLLAAPREDL-NH2 ZElan094R  4H-K(ε-dansyl)KKCAAVLLPVLLAAPC-NH2 ZElan176  5H-K(ε-dansyl)CAAVLLPVLLAAC-NH2 ZElan177  6H-K(ε-dansyl)KKCAAVLLPVLLAC-NH2 ZElan178  7 H-K(ε-dansyl)CAAVLLPVLLC-NH2ZElan179  8 H-K(ε-dansyl)CAAVLLPVLC-NH2 ZElan180  9H-K(ε-dansyl)CAVLLPVLLAAPC-NH2 ZElan181 10 H-K(ε-dansyl)CVLLPVLLAAPC-NH2ZElan182 11 H-K(ε-dansyl)CLLPVLLAAPC-NH2 ZElan183 12H-K(ε-dansyl)CLPVLLAAPC-NH2 ZElan184 13 H-K(ε-dansyl)AAVLLPVLLAAP-NH2ZElan185 14 H-K(ε-dansyl)AAVLLPVLLAA-NH2 ZElan186 15H-K(ε-dansyl)KKAAVLLPVLLA-NH2 ZElan187 16 H-K(ε-dansyl)AAVLLPVLL-NH2ZElan188 17 H-K(ε-dansyl)AAVLLPVL-NH2 ZElan189 18H-K(ε-dansyl)AVLLPVLLAAP-NH2 ZElan190 19 H-K(ε-dansyl)VLLPVLLAAP-NH2ZElan191 20 H-K(ε-dansyl)LLPVLLAAP-NH2 ZElan192 21H-K(ε-dansyl)LPVLLAAP-NH2 ZElan193 22 H-K(ε-dansyl)AAVLLPVLLAAKKKRKA-NH2Zelan204N 23 H-K(ε-dansyl)KKKRKAAAAVLLPVLLA-NH2 ZElanN204 24

[0119] An L-peptide that has an amino acid sequence of SEQ ID NO: 2would be KKAAAVLLPVLLAAP. A composition that comprises ZElan094,comprises an L-peptide of SEQ ID NO: 2, and further comprises both aK(ε-dansyl) group and an amide group.

[0120] The 16 residue hydrophobic peptide ZElan094 (SEQ ID NO: 2) isrelated in sequence to the 12 residue hydrophobic peptide sequenceAAVLLPVLLAAP (SEQ ID NO: 1) (Rojas et al. Nature Biotechnology 16:370,1998). However, the 16 residue ZElan094 differs from the 12 residue SEQID NO: 1 in that it has four additional amino acid residues, KKKA, atthe N-terminus and a blocking amide at the C-terminus. These N-terminusand C-terminus modifications are designed to enhance the solubility andthe in vivo stability of the MTLP, respectively. The NH₂ terminusalanine also may contribute to the alpha helical properties of thepeptide.

[0121] The MTLPs of the present invention include peptides comprisingall of or a fragment of ZElan094 or having at least 4 of the contiguousamino acids of ZElan094. The MTLPs of the present invention also includesequences that are substantially homologous to regions of ZElan094.Preferably these show at least 70%, 80% or 90% identity over anidentical size sequence.

[0122] It is understood that a person in the art can make chemicalchanges in the MTLPs without significantly altering their activity.

[0123] Examples of nucleic acid sequences, which encode the peptidesequences of the MTLPs ZElan094, Felan 094, ZElan 094R, 176-193, 204Nand N204 (SEQ ID NOS: 2-24) are provided in Table 2 (SEQ ID NOS: 25-47).However, due to the degeneracy of nucleotide coding sequences, differentnucleotide sequences, which encode substantially the same amino acidsequence, may be used. That is, a nucleotide sequence, altered bysubstitution of a different codon, can encode a functionally equivalentamino acid to produce a silent change.

[0124] MTLPs may be synthesized using chemical methods (U.S. Pat. Nos.4,244,946, 4,305,872 and 4,316,891; Merrifield et al. J. Am. Chem. Soc.85:2149, 1964; Vale et al. Science 213:1394, 1981; Marki et al. J. Am.Chem. Soc. 103:3178, 1981); recombinant DNA methods (Maniatis, MolecularCloning, a Laboratory Manual, 2d ed. Cold Spring Harbor Laboratory, ColdSpring Harbor N.Y., 1990); viral expression or other methods known tothose skilled in the art.

[0125] Chemical methods include, but are not limited to, solid phasepeptide synthesis. Briefly, solid phase peptide synthesis consists ofcoupling the carboxyl group of the C-terminal amino acid to a resin andsuccessively adding N-alpha protected amino acids. The protecting groupsmay be any known in the art. Before an amino acid is added to thegrowing peptide chain, the protecting group of the previous amino acidis removed (Merrifield J. Am. Chem. Soc. 85:2149 1964; Vale et al.Science 213:1394, 1981; Marki et al. J. Am. Chem. Soc. 103:3178,1981).The synthesized peptides are then purified by methods known in the art.TABLE 2 MTLPs nucleic acid sequence SEQ ID ZElan NO: NO: Sequence 25  94AARAARAARGCNGCNGCNGTNYTNYTNCCNGTNYTN YTNGCNGCNCCN 26 Felan094AARAARAARGCNGCNGCNGTNYTNYTNCCNGTNYTN YTNGCNGCNCCN 27 094RAARAARAARGCNGCNGCNGTNYTNYTNCCNGTNYTN YTNGCNGCNCCNMGNGARGAYYTN 28 176AARAARAARTGYGCNGCNGTNYTNYTNCCNGTNYTN YTNGCNGCNCCNTGY 29 177AARAARAARTGYGCNGCNGTNYTNYTNCCNGTNYTN YTNGCNGCNTGY 30 178AARAARAARTGYGCNGCNGTNYTNYTNCCNGTNYT NYTNGCNTGY 31 179AARTGYGCNGCNGTNYTNYTNCCNGTNYTNYTNTGY 32 180AARTGYGCNGCNGTNYTNYTNCCNGTNYTNTGY 33 181AARTGYGCNGTNYTNYTNCCNGTNYTNYTNGCNGCN CCNTGY 34 182AARTGYGTNYTNYTNCCNGTNYTNYTNGCNGCNCCN TGY 35 183AARTGYYTNYTNCCNGTNYTNYTNGCNGCNCCNTGY 36 184AARTGYYTNCCNGTNYTNYTNGCNGCNCCNTGY 37 185AARGCNGCNGTNYTNYTNCCNGTNYTNYTNGCNGCN CCN 38 186AARGCNGCNGTNYTNYTNCCNGTNYTNYTNGCNGCN 39 187AARAARAARGCNGCNGTNYTNYTNCCNGTNYTNYTN GCN 40 188AARGCNGCNGTNYTNYTNCCNGTNYTNYTN 41 189 AARGCNGCNGTNYTNYTNCCNGTNYTNYTN 42190 AARGCNGTNYTNYTNCCNGTNYTNYTNGCNGCNCCN 43 191AARGTNYTNYTNCCNGTNYTNYTNGCNGCNCCN 44 192 AARYTNYTNCCNGTNYTNYTNGCNGCNCCN45 193 AARYTNCCNGTNYTNYTNGCNGCNCCN 46 204NAARGCNGCNGTNYTNYTNCCNGTNYTNYTNGCNGCN AARAARAARMGNAARGCN 47 N204AARAARAARAARMGNAARGCNGCNGCNGCNGTNYTN YTNCCNGTNYTNYTNGCN

[0126] Preferably, solid phase peptide synthesis is done using anautomated peptide synthesizer such as, but not limited to, an AppliedBiosystems Inc. (ABI) model 431A using the “Fastmoc” synthesis protocolsupplied by ABI. This protocol uses2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) as coupling agent (Knorr et al. Tet. Lett. 30:1927,1989).Syntheses can be carried out on 0.25 mmol of commercially available4-(2′, 4′-dimethoxyphenyl-(9-fluorenyl-ethoxycarbonyl)-aminomethyl)phenoxy polystyrene resin (Rink H. Tet. Lett. 28:3787, 1987). Fmoc aminoacids (1 mmol) are coupled according to the Fastmoc protocol.N-methylpyrrolidone (NMP) is used as solvent, with HBTU dissolved inN,N-dimethylformamide (DMF). The following side chain protected Fmocamino acid derivatives are used: FmocArg(Pmc)OH; FmocAsn(Mbh)OH;FmocAsp(tBu)OH; FmocCys(Acm)OH; FmocGlu(tBu)OH; FmocGln(Mbh)OH;FmocHis(Tr)OH; FmocLys(Boc)OH; FmocSer-(tBu)OH; FmocThr(tBu)OH;FmocTyr(tBu)OH. (Abbreviations: Acm:acetamidomethyl;Boc:tert-butoxycarbonyl; tBu:tert-butyl; Fmoc:9-fluorenylmethoxy-carbonyl; Mbh:4,4′-dimethoxybenzhydryl;Pmc:2,2,5,7,8-pentamethyl-chro-man-6-sulfonyl; Tr:5 trityl.)

[0127] At the end of each synthesis, the amount of peptide is assayed byultraviolet spectroscopy. A sample of dry peptide resin (about 3-10 mg)is weighed, then 20% piperidine in DMA (10 ml) is added. After 30 minsonication, the UV (ultraviolet) absorbance of thedibenzofulvene-piperidine adduct (formed by cleavage of the N-terminalFmoc group) is recorded at 301 nm. Peptide substitution (in mmol/g) iscalculated according to the equation:${Substitution} = \frac{A \times v \times 1000}{7800 \times w}$

[0128] where A is the absorbance at 301 nm, v the ml of 20% piperidinein DMA, 7800 the extinction coefficient (mol/dm³/cm) of thedibenzofulvene-piperidine adduct, and w the mg of peptide resin sample.The N-terminal Fmoc group is cleaved using 20% piperidine in DMA, thenacetylated using acetic anhydride and pyridine in DMA. The peptide resinis thoroughly washed with DMA, CH₂C₁₂ and diethyl ether.

[0129] Methods used for cleavage and deprotection (King et al. Int. J.Peptide Protein Res. 36:255, 1990) include, but are not limited to,treating the air-dried peptide resin with ethylmethyl-sulfide (EtSMe),ethanedithiol (EDT) and thioanisole (PhSMe) for approximately 20 min andadding 95% aqueous trifluoracetic acid (TFA). Approximately 50 ml ofthese reagents are used per gram of peptide resin in a ratio ofTFA:EtSMe:EDT:PhSme (10:0.5:0.5:0.5). The mixture is stirred for 3 h atRT under an N₂ atmosphere, filtered and washed with TFA (2×3 ml). Thecombined filtrate is evaporated in vacuo and anhydrous diethyl ether isadded to the yellow/orange residue. The resulting white precipitate isisolated by filtration. Purification of the synthesized peptides is doneby standard methods including, but not limited to, ion exchange,affinity, sizing column and high performance liquid chromatography,centrifugation or differential solubility.

[0130] Recombinant DNA methods for expressing peptides are well known tothose skilled in the art and include expression in a biological systemincluding, but not limited to a mammalian system, an insect system, aplant system and a viral system (Maniatis, T. Molecular Cloning, ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1990). For example, a MTLP can be expressed by a virus, bya virus fused to a viral coat protein, a viral capsid protein or a viralsurface protein. Further, MTLP-viral protein complexes can be expressedin mammalian hosts or in helper viruses used to produce the virus ofinterest.

[0131] In the production of a gene encoding an extended version of, or afragmentof a full-length peptide, care should be taken to ensure thatthe modified gene remains within the same translational reading frameuninterrupted by translational stop signals in the gene region where thedesired activity is encoded.

[0132] Further, phage display vectors including, but not limited to,bacteriophage M13 or bacteriophage Fd can be modified to express a MTLPfused to the gene III protein product or gene VII protein product of thebacteriophage. A library of sequences coding for MTLPs or potentialMTLPs can be created including, but not limited to, alanine scanpositional mutants, successive random positional scanning mutants andsequences derived therefrom as, for example, those shown in Table 1, canbe cloned in-frame to either gene II or gene VII of the bacteriophage.The phage display library can then be screened to identify new MTLPshaving enhanced ability to transport active agents or active particlesacross membranes.

[0133] Chimeric or fusion peptides include, without limitation, thosecomprising a MTLP or multiple repeats thereof, preferably consisting ofat least one domain or motif of the full-length peptide sequence or aportion thereof joined at its amino-terminus, at its carboxy-terminus orat an internal site via a peptide bond to an amino acid sequence of adifferent peptide. Methods for producing chimeric peptides include, butare not limited to, recombinant expression of a nucleic acid includingthe MTLP coding sequence joined in-frame to the coding sequence of adifferent peptide. Using methods known in the art, the nucleic acidsequences encoding the desired amino acid sequences are ligated to eachother in the proper order and the chimeric product is expressed. Forexample, chimeric genes comprising portions of MTLP nucleic acid fusedto any heterologous protein-encoding nucleic acid may be constructed.Alternatively, chimeric MTLPs may be synthesized using techniquesincluding, but not limited to, a peptide synthesizer. Opioid peptidesinclude those contained in the corticotropin-lipoportein precursor, theproenkaphalin A precursor, and the beta-neoendorphin-dynorphinprecursor, as follows: COLI_HUMAN (P01189) Corticotropin-lipotropinprecursor (Pro-opiomelanocortin) (POMC) Contains: NPP; Melanotropingamma (Gamma-MSH); Corticotropin (Adrenocorticotropic hormone) (ACTH);Melanotropin alpha (Alpha-MSH); Corticotropin-like intermediary peptide(CLIP); Lipotropin beta (Beta-LPH); Lipotropin gamma (Gamma-LPH);Melanotropin beta (Beta-MSH); Beta-endorphin; and Met- enkephalin].PENK_HUMAN (P01210) Proenkephalin A precursor contains:Met-enkephalin;and Leu-enkephalin. NDDB_HUMAN (P01213)Beta-neoendorphin-dynorphin precursor (Proenkephalin B)(Preprodynorphin) contains: Beta-neoendorphin; Dynorphin;Leu-Enkephalin; Rimorphin; and Leumorphin]. Their amino acid sequencesas obtained from the SWISSPROT database are as follows: LOCUS COLI_HUMAN267 aa linear PRI 01-MAR-2002 DEFINITION Corticotropin-lipotropinprecursor (Pro-opiomelanocortin) (POMC) [Contains: NPP; Melanotropingamma (Gamma-MSH); Corticotropin (Adrenocorticotropic hormone) (ACTH);Melanotropin alpha (Alpha-MSH); Corticotropin-like intermediary peptide(CLIP); Lipotropin beta (Beta-LPH); Lipotropin gamma (Gamma-LPH);Melanotropin beta (Beta-MSH); Beta-endorphin; Met-enkephalin]. ACCESSIONP01189 PID g116880 VERSION P01189 GI:116880 DBSOURCE swissprot: locusCOLI_HUMAN, accession P01189; SOURCE human. Region 27..102 = NPP Region77..87 =“MELANOTROPIN GAMMA.” Region 105..134 =“?” Region 138..150=MELANOTROPIN ALPHA.” Region 138..176 =“CORTICOTROPIN.” Region 156..176=“CORTICOTROPIN-LIKE INTERMEDIARY PEPTIDE.” Region 179..234 =“LIPOTROPINGAMMA.” Region 179..267 =“LIPOTROPIN BETA.” Region 217..234=“MELANOTROPIN BETA.” Region 237..241 =“MET-ENKEPHALIN.” Region 237..267=“BETA-ENDORPHIN.” ORIGIN 1 mprsccsrsg alllalllqa smevrgwcle ssqcqdlttesnllecirac kpdlsaetpm 61 fpgngdeqpl tenprkyvmg hfrwdrfgrr nssssgssgagqkredvsag edcgplpegg 121 peprsdgakp gpregkrsys mehfrwgkpv gkkrrpvkvypngaedesae afplefkrel 181 tgqrlregdg pdgpaddgag aqadlehsll vaaekkdegpyrmehfrwgs ppkdkryggf 241 mtseksqtpl vtlfknaiik naykkge--------------------------------------------------------------------------LOCUS PENK_HUMAN 267 aa linear PRI 15-JUL-1999 DEFINITION PROENKEPHALINA PRECURSOR [CONTAINS: MET-ENKEPHALIN; LEU-ENKEPHALIN]. ACCESSION P01210PID g129770 VERSION P01210 GI:129770 DBSOURCE swissprot: locusPENK_HUMAN, accession P01210; SOURCE human. Region 100..104=“MET-ENKEPHALIN 1.” Region 107..111 =“MET-ENKEPHALIN 2.” Region136..140 =“MET-ENKEPHALIN 3.” Region 186..193=“MET-ENKEPHALIN-ARG-GLY-LEU.” Region 210..214 =“MET-ENKEPHALIN 4.”Region 230..234 =“LEU-ENKEPHALIN.” Region 261..267 =“MET-ENKEPHALIN-ARG-PHE.” ORIGIN 1 marfltlctw llllgpglla tvraecsqdc atcsyrlvrpadinflacvm ecegklpslk 61 iwetckellq lskpelpqdg tstlrenskp eeshllakryggfmkryggf mkkmdelypm 121 epeeeangse llakryggfm kkdaeeddsl anssdllkelletgdnrers hhqdgsdnee 181 evskryggfm rglkrspqle deakelqkry ggfmrrvgrpewwmdyqkry ggflkrfaea 241 lpsdeegesy skevpemekr yggfmrf //---------------------------------------------------------------- LOCUSPENK_HUMAN 267 aa linear PRI 15-JUL-1999 DEFINITION PROENKEPHALIN APRECURSOR [CONTAINS: MET-ENKEPHALIN LEU-ENKEPHALIN]. ACCESSION P01210PID g129770 VERSION P01210 GI:129770 DBSOURCE swissprot: locusPENK_HUMAN, accession P01210; SOURCE human. Region 100..104=“MET-ENKEPHALIN 1.” Region 107..111 =“MET-ENKEPHALIN 2.” Region136..140 =“MET-ENKEPHALIN 3.” Region 186..193=“MET-ENKEPHALIN-ARG-GLY-LEU.” Region 210..214 =“MET-ENKEPHALIN 4.”Region 230..234 =“LEU-ENKEPHALIN.” Region 261..267=“MET-ENKEPHALIN-ARG-PHE.” ORIGIN 1 marfltlctw llllgpglla tvraecsqdcatcsyrlvrp adinflacvm ecegklpslk 61 iwetckellq lskpelpqdg tstlrenskpeeshllakry ggfmkryggf mkkmdelypm 121 epeeeangse ilakryggfm kkdaeeddslanssdllkel letgdnrers hhqdgsdnee 181 evskryggfm rglkrspqle deakelqkryggfmrrvgrp ewwmdyqkry ggflkrfaea 241 lpsdeegesy skevpemekr yggfmrf //----------------------------------------------------------

[0134] The SwissProt database records for accession numbers P01189,P01210, and P01213 as they appeared on Apr. 17, 2002 are incorporatedherein by reference in their entireties.

[0135] Additional opioid peptides include, but are not limited to:

[0136] boc-Tyr-Tic

[0137] cy-[Tyr-Tic]

[0138] Tyr-D-Tic

[0139] Tyr-D-Tic-NH2

[0140] Tyr-cy-[D-Cys-Phe-D-Pen]

[0141] Tyr-D-Tic-Phe-Phe-NH2

[0142] Tyr-Tic-Phe-Phe

[0143] Tyr-cy-[D-Pen-Ala-Phe-D-Pen]

[0144] Tyr-cy-[D-Pen-D-Ala-Phe-D-Pen]

[0145] Tyr-cy-[D-Pen-Gly-Phe-D-Pen]

[0146] Tyr-cy-[D-Pen-Ser-Phe-Pen]

[0147] Tyr-D-Pen-Gly-Phe-DMPT

[0148] Tyr-D-Ala-Gly-Phe-D-Leu

[0149] Tyr-D-Nle-Gly-Phe-NleS

[0150] Tyr-Gly-Gly-Phe-Leu

[0151] Tyr-Gly-Gly-Phe-Met

[0152] Tyr-D-The-Gly-Phe-Leu-Thr

[0153] N,N-diallyl-(O-t-butyl)-Tyr-Aib-Aib-Phe-Leu-O-Me

[0154] Tyr-cy-[D-Pen-Gly-Phe-D-Pen]-Nle-Gly-NH2

[0155] Tyr-Gly-Gly-Phe-NH-NH-Phe-Gly-Gly-Tyr

[0156] [Leu]-enkephalin

[0157] Where the following abbreviations are used:

[0158] Pen—penicilamine

[0159] Nle—nor-leucine (CH3-CH2-CH2-CH2-CH-(NH2)COOH)

[0160] NleS—CH3-CH2-CH2-CH2-CH-(NH2)SO3H

[0161] Tic—tetrahydroisoquinoline-3-carboxylic acid

[0162] Aib—alpha-aminoisobutryric acid

[0163] cy—cyclo

[0164] MTLPs may be linked to other molecules including, but not limitedto, detectable labels, adsorption facilitating molecules, toxins orsolid substrata by methods including, but not limited to, the use ofhomobifunctional and heterobifunctional cross-linking molecules(Carlsson et al. Biochem. J. 173:723, 1978; Cumber et al. Methods inEnzymology 112:207, 1978; Jue et al. Biochem. 17:5399,1978; Sun et al.Biochem. 13:2334, 1974; Blattler et al. Biochem. 24:1517, 1985; Liu etal. Biochem. 18:690, 1979; Youle and Neville Proc. Natl. Acad. Sci. USA77:5483,1980; Lerner et al. Proc. Natl. Acad. Sci. USA 78:3403.1981;Jung and Moroi Biochem. Biophys. Acta 761:162 1983; Caulfield et al.Biochem. 81:7772, 1984; Staros Biochem. 21:3950, 1982; Yoshitake et al.Eur. J. Biochem. 101:395, 1979; Yoshitake et al. J. Biochem. 92:1413,1982; Pilch and Czech J. Biol. Chem. 254:3375, 1979; Novick et al. J.Biol. Chem. 262:8483. 1987; Lomant and Fairbanks J. Mol. Biol. 104:243,1976; Hamada and Tsuruo Anal. Biochem. 160:483,1987; Hashida et al J.Applied Biochem. 6:56,1984; Means and Feeney Bioconjugate Chem.1:2,1990).

[0165] MTLPs may be used as immunogens to generate antibodies whichimmunospecifically bind the immunogen. Such antibodies include, but arenot limited to polyclonal, monoclonal, chimeric, single chain Fabfragments, F(ab′)₂ fragments and Fab expression libraries. Uses of suchantibodies include, but are not limited to, localization, imaging,diagnosis, treatment and treatment efficacy monitoring. For example,antibodies or antibody fragments specific to a domain of a MTLP, such asa dansyl group or some other epitope introduced into the peptide, can beused to identify the presence of the MTLP, to bind the MTLP to thesurface of a particle, to quantitate the amount of the MTLP on aparticle, to measure the amount of the MTLP in a physiological sample,to immunocytochemically localize the MTLP in a cell or tissue sample, toimage the MTLP after in vivo administration and to purify the MTLP byimmunoaffinity column chromatography.

[0166] The functional activity of a MTLP can be determined by suitablein vivo or in vitro assays known to those skilled in the art. Theseinclude, but are not limited to, immuno-, immunoradiometric-,immunodiffusion- and immunofluorescence assays and to western blotanalysis.

[0167] A MTLP functions to target an active agent or an active particleto a cell, intracellular compartment, or cell layer and to enhance theuptake of the active agent or of the active particle into a cell, intoand out of an intracellular compartment and across a cell layer. Cellsinclude, but are not limited to, epithelial, endothelial and mesothelialcells, unicellular organisms and plant cells. Cell layers includeepithelial, endothelial and mesothelial cell layers such as, but notlimited to, the gastrointestinal tract, pulmonary epithelium, bloodbrain barrier and vascular endothelium. Preferably the cell is anepithelia, cell and the cell layer is an epithelial cell layer. Mostpreferably, the cell is a GIT epithelial cell and the cell layer is theGIT epithelial cell layer. Intracellular compartments include, but notlimited to, nuclear, mitochondrial, endoplasmic reticular and endosomalcompartments. MTLPs can be used to enhance the uptake of an active agentor active particle that regulates or directs intra-cellular trafficking.Further, MTLPs can be used to enhance intracellular gene delivery. Thatis, a gene or plasmid DNA is encapsulated or complexed within a cationiclipid polymer system and the surface of the cationic lipid polymersystem is complexed with an MTLP or with a targeting peptide.Alternatively, a plasmid DNA is condensed, the condensate is complexedwith cationic lipids and the surface of the cationic lipids is complexedwith an MTLP or with a targeting peptide.

[0168] Methods used to complex a MTLP to an active agent (MTLP-activeagent complex) include, but are not limited to, covalent coupling of aMTLP and an active agent, either directly or via a linking moiety,noncovalent coupling of a MTLP and an active agent and generation of afusion protein, wherein a MTLP is fused in-frame to an active agentincluding, but not limited to a therapeutic protein.

[0169] Methods used to complex a MTLP to an active agent loaded particle(MTLP-active particle complex) include, but are not limited to,adsorption to the active particle, noncovalent coupling to the activeparticle; covalent coupling, either directly or via a linker, to theactive particle, to the polymer or polymers used to synthesize theactive particle, to the monomer or monomers used to synthesize thepolymer; and, to any other component comprising the active particle.Further, MTLPs can be complexed to a slow-release (controlled release)particle or device (Medical Applications of Controlled Release, Langer &Wise (eds.), CRC Press, Boca Raton, Fla., 1974; Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York, 1984; Ranger et al. J. Macromol. Sci. Rev.Macromol. Chem. 23:61, 1983; Levy et al. Science 228:190, 1985; Duringet al. Ann. Neurol. 25:351, 1989; Howard et al. J. Neurosurg. 71:1051989).

[0170] Methods used for viral based gene delivery systems include, butare not limited to, vectors modified at the nucleic acid level toexpress a MTLP on the surface of a viral particle and mammalian cells orhelper viruses, which express MTLP-virus fusion proteins that areincorporated into a viral vector.

[0171] The present invention also provides pharmaceutical formulations,comprising a therapeutically effective amount of a MTLP-active agentcomplex or of a MTLP-active particle complex and a pharmaceuticallyacceptable carrier (Remington's Pharmaceutical Sciences by E. W.Martin). The term “pharmaceutically acceptable” includes, but is notlimited to, carriers approved by a regulatory agency of a country or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the MTLP-active agent complex or the MTLP-activeparticle complex is administered. Such pharmaceutical carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Water is a preferred carrier whenthe pharmaceutical formulation is administered orally. Saline solutionsand aqueous dextrose and glycerol solutions can also be employed asliquid carriers, particularly for injectable solutions. Suitablepharmaceutical excipients include starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The formulation, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions include, but are notlimited to, solutions, suspensions, emulsion, tablets, pills, capsules,powders and sustained-release formulations. The formulation can be asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers including,but not limited to, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose and magnesiumcarbonate. Such formulations will contain a therapeutically effectiveamount of the active agent or of the active agent loaded into aparticle, together with a suitable amount of carrier so as to providethe form for proper administration to an individual in need of theactive agent.

[0172] Any route known in the art may be used to administer aMTLP-active agent complex or a MTLP-active particle complex, includingbut not limited, to oral, nasal, topical, mucosal, intravenous,intraperitoneal, intradermal, intrathecal, intramuscular, transdermaland osmotic. Preferably, administration is oral, wherein the MTLPenhances uptake of the active agent into a GIT epithelial cell andacross the GIT epithelium into the circulation. The precise amount ofactive agent to be administered for the diagnosis, prevention ortreatment of a particular pathological condition will depend on thepathological disorder, the severity of the pathological disorder, theactive agent used and the route of administration. The amount of activeagent to be administered and the schedule of administration can bedetermined by the practitioner using standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal ranges for active agent administration.

[0173] The following examples will serve to further illustrate thepresent invention without, at the same time, however, constituting anylimitation thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLE 1

[0174] Peptide Synthesis

[0175] The membrane translocating peptides ZElan094, 204N and 204 andthe targeting peptides HAX42, PAX2, P31 and Sni34 (U.S. patentapplications Ser. Nos. 09/079,819, 09/079,723 and 09/079,678) weresynthesized chemically using a fmoc synthesis protocol (Anaspec, Inc.,San Jose, Calif.). A dansyl group was added at the N-terminus of eachsequence in order to enable the detection of the peptide withanti-dansyl antibody (Table 1).

[0176] The physical characteristics of Zelan094 (SEQ ID NO: 2) are shownin Table 3. TABLE 3 Physical characteristics of ZElan 094 (SEQ ID NO: 2)Mass (M+H+): 1838.03 Solubility 1 mg/ml water Appearance white powderHPLC purity >95% Kyle-Doolittle Hydropathy Plot

EXAMPLE 2 Preparation of MTLP-active Particle Complexes and of TargetingPeptide-Active Particle Complexes

[0177] Active particles were prepared from a polymer using acoacervation method. Preferably, particle size is between about 5 nm and750 μm, more preferably between about 10 nm and 500 μm and mostpreferably between about 50 nm and 800 nm. MTLPs or targeting peptideswere complexed to the particles using various methods known to thoseskilled in the art.

[0178] The following is a general method for preparation of coacervatedparticles.

[0179] Phase A

[0180] A polymer agent, a surface-active agent, a surface-stabilizingagent, a surface-modifying agent or a surfactant is dissolved in water(A). Preferably the agent is a polyvinyl alcohol (hereinafter “PVA”) ora derivative thereof having a % hydrolysis of about 50-100 and amolecular weight range of about 500-500,000 kDa. More preferably theagent is a PVA having a % hydrolysis of 80-100 and a molecular weightrange of about 10,000-150,000 kDa. The mixture (A) is stirred under lowshear conditions at 10-2000 rpm and, more preferably, at 100-600 rpm.The pH and ionic strength of the solution may be modified using salts,buffers or other modifying agents. The viscosity of the solution may bemodified using polymers, salts, or other viscosity modifying agents.

[0181] Phase A may include agents such as, but not limited to,emulsifying agents, detergents, solubilizing agents, wetting agents,foaming agents, antifoaming agents, flocculents and defloculents.Examples include, but are not limited to, anionic surface agents such assodium dodecanoate, sodium dodecyl-(lauryl)sulphate, sodiumdioctyl-sulphosuccinate, cetostearyl alcohol, stearic acid and its saltssuch as magnesium stearate and sodium stearate, sodium dodecyl-benzenesulphonate, sodium cholate triethanolamine; cationic surface agents suchas hexadecyl trimethyl ammonium bromide (cetrimide), dodecyl pyridiniumiodide, dodecyl pyridinium chloride; non-ionic surface agents such ashexaoxyethylene monohexadecyl ether, polysorbates (Tweens), sorbitanesters (Spans), Macrogol ethers, Poloxalkols (Poloxamers), PVA, PVP,glycols and glycerol esters, fatty alcohol poly glycol ethers, dextrans,higher fatty alcohols; and, amphoteric surface agents such as N-dodecylalanine, lecithin, proteins, peptides, polysaccharides, semisyntheticpolysaccharides, sterol-containing substances, and finely divided solidssuch as magnesium hydroxide and montmorillonite clays.

[0182] Phase B

[0183] A polymer is dissolved in a water miscible organic solvent toform the organic phase (B). Preferably the organic phase is anacetone-ethanol mixture in ratios from 0:100 acetone:ethanol to 100:0acetone:ethanol depending upon the polymer used. Other polymers,peptides, sugars, salts, natural-polymers, synthetic polymers or otheragents may be added to the organic phase (B) to modify the physical andchemical properties of the resultant particle product.

[0184] The polymers may be soluble, permeable, impermeable,biodegradable or gastroretentive. They may be a mixture of natural orsynthetic polymers and copolymers. Such polymers include, but are notlimited to, polylactides, polyglycolides, DL, L and D forms ofpoly(lactide-coglycolides) (PLGA), copolyoxalates, polycaprolactone,polyester-amides, polyorthoesters, polyanhydrides,polyalkyl-cyano-acrylates, polyhydroxy-butyrates, polyurethanes,albumin, casein, citosan derivatives, gelatin, acacia, celluloses,polysaccharides, alginic acid, polypeptides and the like, copolymersthereof, mixtures thereof, enantiomeric forms thereof, stereoisomersthereof and any MTLP conjugate thereof. Synthetic polymers include, butare not limited to, alkyl celluloses, hydroxyalkyl celluloses, celluloseethers- cellulose esters, nitrocelluloses, acrylic and methacrylic acidsand esters thereof, dextrans, polyamides, polycarbonates, polyalkylenes,polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates,polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinylhalides, polyvinylpyrrolidones, polysiloxanes, polyurethanes andcopolymers thereof.

[0185] Phase C

[0186] Phase B is stirred into phase A at a continuous rate. Solvent isevaporated, preferably by increasing the temperature over ambient and/orby using a vacuum pump. The resultant particles are in the form of asuspension (C) An active agent may be added into phase A or into phaseB. Active agent loading may be in the range 0-90% w/w. An MTLP or atargeting peptide may be added into phase C. MTLP and targeting peptideloading may be in the range 0-90% W/W.

[0187] Phase D

[0188] The particles (D) are separated from the suspension (C) usingstandard colloidal separation techniques including, but not limited to,centrifugation at high ‘g’ force, filtration, gel permeationchromatography, affinity chromatography or charge separation. The liquidphase is discarded and the particles (D) are re-suspended in a washingsolution such as, but not limited to, water, salt solution, buffer ororganic solvent. The particles are separated from the washing liquidusing standard colloidal separation techniques and are washed two ormore times. A MTLP or targeting peptide may be used to wash theparticles or, alternatively, may be dissolved in the final wash. Theparticles are dried.

[0189] A secondary layer of polymers, peptides sugars, salts, naturaland/or biological polymers or other agents may be deposited onto thepreformed particulate core by any suitable method known in the art. Thedried particles can be further processed by, for example, tableting,encapsulating or spray drying. The release profile of the particlesformed may be varied from immediate to controlled or delayed releasedepending on the formulation used and/or desired.

EXAMPLE 3 Bovine Insulin Loaded-MTLP Coated Nanoparticles—MTLP Added inthe Final Wash

[0190] Fast acting bovine insulin (28.1 lU/mg) was incorporated intopolylactide-co-glycolide (PLGA, Boehringer Ingelheim, Indianapolis,Ind.) at a theoretical loading of 300 IU of insulin/210 mg ofnanoparticles and the nanoparticles were coated with the dansylatedZElan094 (SEQ ID NO: 2). COMPONENT AMOUNT PLGA RG504H (Lot # 250583) 2 gAcetone 45 mls Ethanol 5 mls PVA (5% w/v) (13-23 kDa, 98% hydrolysis)400 mls Bovine Insulin (Lot # 86HO674) 100 mg ZElan094 (SEQ. ID NO: 2)10 mg/50 ml dH20

[0191] Preparation:

[0192] 1. Water was heated to near boiling, PVA was added to 5% w/v andthe solution was stirred until cool (phase A).

[0193] 2. Acetone and ethanol were mixed to form the organic phase(phase B).

[0194] 3. PLGA was added to the acetone and ethanol (step 2) anddissolved by stirring (phase B).

[0195] 4. An IKA™ reactor vessel was set at 25° C. Phase A (step 1) wasadded into the reactor vessel and stirred at 400 rpm.

[0196] 5. Bovine insulin was added into the stirring phase A (step 4).

[0197] 6. Using clean tubing and a green needle, phase B (step 3) wasslowly dripped into the stirring solution (step 5) using a peristalticpump set at 40.

[0198] 7. The solvent was evaporated by opening the IKA™ reactor vesselports and stirring overnight at 400 rpm to form a suspension (phase C).

[0199] 8. The suspension, phase C (step 7) was centrifuged in a XL90centrifuge at 12,500 to 15,000 rpm for 25 to 40 minutes at 4° C.

[0200] 9. The supernatant was discarded, the particle “cake” broken up,and the particles (phase D) washed twice in 200 ml of dH₂ 0 bycentrifugation in an XL90 centrifuge at 12,500 to 15,000 rpm for 10-15minutes at 4° C. The dansylated ZElan094 (SEQ ID NO: 2) was added intothe final wash.

[0201] 10. The supernatant was decanted, the ‘cake’ broken up and theparticles dried in a vacuum oven. The dried particles were ground,placed in a securitainer and analyzed. Insulin loading was 5% or 50 mginsulin/g particles. Insulin potency, determined in HPLC, was 51.4 mg/g.Scanning electron microscopy showed discrete, reasonably sphericalparticles of about 300-400 nm in diameter.

EXAMPLE 4 Bovine Insulin Loaded-MTLP Coated Nanoparticles—MTLP Added toPhase C

[0202] Fast acting bovine insulin (28.1 lU/mg) was incorporated intoPLGA nanoparticles at a theoretical loading of 300 IU of insulin/210 mgof nanoparticles and the nanoparticles were coated with the MTLPZElan094 (SEQ ID NO: 2). COMPONENT AMOUNT PLGA RG504H (Lot # 250583) 2 gAcetone 45 mls Ethanol 5 mls PVA (5% w/v) (13-15 kDa, 98% hydrolysis)400 mls Bovine Insulin (Lot #. 86HO674) 100 mg ZElan094 (SEQ. ID NO: 2)10 mg/50 ml dH20

[0203] Preparation:

[0204] See steps 1-4 of Example 3.

[0205] Step 5.

[0206] Insulin and ZElan094 were added to the stirring PVA solution.

[0207] See steps 6-9 of Example 3.

[0208] The particles (step 9) were ground, placed in a securitainer andanlayzed.

EXAMPLE 5 Bovine Insulin Loaded-MTLP Coated Nanoparticles—MTLP Added 1Hour Prior to Centrifugation

[0209] Fast acting bovine insulin (28.1 Iu/mg) was incorporated intoPLGA nanoparticles at a theoretical loading of 300 IU of insulin/210 mgof nanoparticles and the nanoparticles were coated with dansylatedZElan094 (SEQ ID NO: 2). COMPONENT AMOUNT PLGA RG504H (Lot # 250583) 2 gAcetone 45 mls Ethanol 5 mls PVA (5% w/v) (13-15 kDa, 98% hydrolysis)400 mls Bovine Insulin (Lot #. 86HO674) 100 mg ZElan094 (SEQ. ID NO: 2)10 mg/50 ml dH20

[0210] Preparation.

[0211] See steps 1-7 of Example 3.

[0212] Step 8.

[0213] ZElan094 was added to the stirring particle suspension. After 1hr, the suspension was centrifuged at 12,500-14,000 rpm for 20 to 40 minat 4° C.

[0214] See steps 9-10 of Example 3.

EXAMPLE 6 Bovine Insulin Loaded-MTLP Nanoparticles—MTLP ConjugatedPolymer

[0215] Fast acting bovine insulin is incorporated into PLGA-dansylatedZElan094 (SEQ ID NO: 2) conjugate nanoparticles at a theoretical loadingof 300 IU of insulin/210 mg of nanoparticles as follows.

[0216] Component

[0217] PLGA RG504H (Lot# 250583)

[0218] RG504H-ZElan094 (SEQ ID NO: 2) conjugate

[0219] Acetone

[0220] Ethanol

[0221] PVA (5%w/v) (13-15 kDa, 98% hydrolysis)

[0222] Bovine Insulin

[0223] Preparation is as in steps 1-10 of Example 3, except that in step3 RG504H and RG504H-ZElan094 conjugate are added to phase B (step 2).

EXAMPLE 7 Bovine Insulin Loaded-Target Peptide Coated Nanoparticles

[0224] Fast acting bovine insulin (28.1 lU/mg) was incorporated intoPLGA nanoparticles at a theoretical loading of 300 IU of insulin/210 mgof nanoparticles and the nanoparticles were coated with the targetingpeptides dansylated ZElan 011, 055, 091, 101, 104, 128, 129 and 144(U.S. patent application Ser. Nos. 09/079,819, 09/079,723 AND09/079,678). COMPONENT AMOUNT PLGA RG504H (Lot # 250583) 2 g Acetone 45ml Ethanol 5 ml PVA (5% w/v) (13-15 kDa, 98% hydrolysis) 400 ml BovineInsulin (Lot #. 86HO674) 100 mg ZElan011, 055, 091, 101, 104, 128, 129and 144 10 mg/50 ml dH20

[0225] (U.S. patent application Ser. Nos. 09/079,819, 09/079,723 AND09/079,678, AND PCT APPLICATION PCT/US98/10088, PUBLISHED AS WO98/51325))

[0226] Preparation:

[0227] See steps 1-10 of Example 3.

[0228] Insulin loading was 5% or 50 mg insulin/g particles.

EXAMPLE 8

[0229] Animal Studies

[0230] In vivo oral insulin bioavailability from MTLP-insulin particlecomplexes (Example 3) and from targeting peptide-insulin particlecomplexes (Example 7) were assessed in the open loop rat model.

[0231] Fifty-nine Wistar rats (300-350 g) were fasted for 4 hours andwere anaesthetized by intramuscular injection of 0.525 ml of ketamine(100 mg/ml)+0.875 ml of acepromazine maleate-BP (2 mg/ml) 15 to 20minutes prior to administration of MTLP-insulin particle complexes or oftargeting peptide-insulin particle complexes. The rats were divided into9 groups, each group containing 6 or 7 animals. Approximately 200 mg ofMTLP-insulin (300 IU) particle complexes, suspended in 1.5 ml of PBS,were injected intra-duodenally at 2-3 cm below the pyloris of each of 6rats (Group 5). Approximately 200 mg of targeting peptide-insulin (300IU) particle, complexes, suspended in 1.5 ml of PBS, were injectedintra-duodenally at 2-3 cm below the pyloris of each of 6-7 rats (Groups1-4 and 6-9). The study groups are( shown in Table 4. TABLE 4 StudyGroups GROUP # # OF RATS PEPTIDE ZELAN NO SEQ ID NO^(a): 1 6 HAX42 09150 2 7 PAX2 144 53 3 7 PAX2 129 54 4 6 P31 101 52 5 6 MTLP 094 48 6 7PAX2 128 55 7 7 PAX2 104 56 8 7 HAX42 011 49 9 7 PAX2 055 51

[0232] Systemic blood was sampled from the tail vein (0.4 ml) of eachrat at 0 minutes and at 15, 30, 45, 60 and 120 minutes afterintra-duodenal administration of the ZElan094-insulin particle complexesor of the targeting peptide-insulin particle complexes. Blood glucose ineach sample was measured using a Glucometer (Bayer; 0.1 to 33.3μm/mol/L). The blood was centrifuged and the plasma was retained. Plasmainsulin was assayed in duplicate using a Phadeseph RIA Kit (Pharmacia,Piscataway, N.J.; 3 to 240 μU/ml).

[0233]FIG. 2 shows the plasma insulin levels following intra-duodenaladministration of ZElan094-insulin particle complexes (Group 5) and oftargeting peptide ZElan091l-(Group 1), 144-(Group 2), 129-(Group 3),101-(Group 4), 128-(Group 6), 104-(Group 7) and 011-(Group 8) insulinparticle complexes. As shown in FIG. 2, during the 60 minutes followingintra-duodenal administration, ZElan094-insulin particles complexesprovided the most potent enhancement of insulin delivery followed bytZElan055-, 129- and 094-, 101-, 128-, 091- and 144, and 011-insulinparticle complexes. These data show that the plasma insulin levelsobtained using MTLP-insulin particle complexes, were greater than thoseobtained using the targeting peptide-insulin particle complexes.

[0234] To ensure that the insulin delivered from the MTLP-insulinparticle complexes and from the targeting peptide-insulin particlecomplexes was bioactive, blood glucosea levels were measured. As shownin FIG. 3, during the 20 minutes following intra-duodenaladministration, blood glucose levels fell from between about 6.0-9.5mmol/L to about 4.5-7.0 mmol/L and remained significantly below controlvalues (PBS) for at least 60 minutes. There was no significantdifferences in blood glucose levels among the animals receiving theMTLP-insulin particle complexes and the animals receiving the targetingpeptide-insulin particle complexes at 60 minutes and at 120 minutes;.These data show that insulin delivered from the dansylatedZElan094-insulin particle complexes and from the dansylated Zelan011,055, 091, 144, 129, 101, 129, 128 and 104-insulin particle complexesremained bioactive. Further, these data show that insulin delivered fromMTLP-insulin particle complexes enabled a significant and long lastingdecrease in blood glucose levels.

EXAMPLE 9 Preparation of DNA Containing Liposomes and of DNA ContainingMTLP Coated Liposomes

[0235] DNA containing liposomes and DNA containing MLTP coated liposomeswere, prepared as follows:

[0236] Solution 1

[0237] Twelve nmol lipofectamine (Gibco BRL, Rockville, Md.), ±0.6 mg ofprotamine sulphate, was prepared in a final volume of 75 ml optiMEM.

[0238] Solution 2

[0239] One mg of pHM6lacZ DNA (Boehringer Mannheim) was prepared in afinal volume of 75 ml optiMEM. The reporter plasmid pHM6lacZ containsthe lacZ gene, which codes for bacterial β-galactosidase.

[0240] Solution 3

[0241] Solution 1 and Solution 2 were combined and incubated for 15minutes at RT to enable complex formation.

[0242] Solution 4

[0243] ZElan094, 204N or 204 (SEQ ID Nos: 2, 23, 24) were added toSolution 3 to a final concentration of 100 mM and incubated for 5minutes at RT. Six-hundred ml of optiMEM was added and the solution wasmixed gently.

[0244] The DNA containing liposomes and the DNA containing MTLP coatedliposom a complexes were analyzed in scanning electron microscopy (SCM)or in transmission electron microscopy (TEM) to confirm complex liposomeformation and by zeta potential analysis to confirm surface chargeproperties.

EXAMPLE 10 Delivery of DNA From Liposomes and from MTLP-Liposomes intoCaco-2 Cells

[0245] DNA delivery into Caco-2 cells from liposomes and from MTLPcoated liposomes was calculated as β-galactosidase expression per mg oftotal protein in the, cell supernatant. β-galactosidase expression wasdetermined using the Boehringer Mannheim chemiluminescence kit. Proteinwas determined using the Pierce Micro bichinconate (BCA) protein assay.

[0246] Caco-2 cells were plated at 1×10⁵ cells/well in 1 ml of culturemedia and incubated at 37° C. in 5% CO₂ overnight. The cells were washedtwice in 0.5 ml of optiMEM. ZElan094, 204N or 204 (SEQ ID NOS: 2, 23,24) (Solution 4, Example 9) were each added to triplicate wells (250μl/well) of the washed cells and incubated for 4 h at 37° C. After 4 h,250 μl of optiMEM containing 2×fetal calf serum was added and the cellswere incubated for an additional 20 h at 37° C. At 24 hpost-transfection, the cells were lysed with Boehringer Mannheim LysisBuffer. The lysate was centrifuged for 2 min at 14,000 rpm in anEppendorf Centrifiguge and the supernatant was collected.

[0247] Table 5 shows relative β-galactosidase expression per mg of totalprotein using ZElan094, ZElan204N and ZElan204 (SEQ ID NOS: 2, 23, 24)coated liposomes as the DNA delivery particles. TABLE 5 β-galactosidaseexpression in Caco-2 cells EXPERIMENTS 1 2 Lipofectamine + DNA (control)100% 100% Lipofectamine + DNA + protamine (control)  90% 162%Lipofectamine + DNA + protamine + ZElan094 387% 260% Lipofectamine +DNA + protamine + ZElan204N 495% 217% Lipofectamine + DNA + protamine +ZElanN204 176% 122%

[0248] The MLTPs ZElan094, 204N and N204 (SEQ ID NOS: 2, 23 and 24)coated liposomes delivered more DNA into the Caco-2 cells than did thelipofectamine+DNA and lipofectamine+DNA+protamine control liposomes.Moreover, as indicated by b-galactosidase expression, the ZElan094derivative ZElan204N, which is modified at the C-terminus by theaddition of a nuclear localisation sequence (NLS), was most effective inenhancing both delivery of DNA into and expression of DNA within Caco-2cells. The MTLP ZElan094 and its derivatives, in combination withcationic lipids and DNA condensing agents, enhanced both the targetingof genes to cells and the subsequent uptake of the genes by the cells.

[0249] As MTLPs enhance uptake of both active-agents andactive-particles into cells, MTLPs including, but not limited to,ZElan094 and ZElan 204N, can be used as coating agents on polymer basedparticle systems and on liposome based particle systems as active agentand active particle delivery systems. Further, MTLPs also can be used ascoating agents on viral vector based particle systems including, but notlimited to, adenovirus, adeno-associated virus, lentivirus, and vacciniavirus. In such systems, the virus itself may code for the MTLP, whereinthe DNA sequence coding for the MTLP has been cloned in frame to one ormore genes which code for one or more, viral capsid protein or for oneor more viral surface proteins. Alternatively, the surface, of the virusused for gene delivery may be modified with a MTLP following virus;production and purification from a cell including, but not limited to, amammalian cell.

EXAMPLE 11 Effects of MTLPs and of the Targeting Peptides on SubstrateTransport Across a Cell Layer

[0250] The effect of the MTLPs ZElan094, ZElan178 and ZElan187 (SEQ IDNOS: 2, 7 and 16) and of the targeting peptide ZElan022 (U.S. patentapplication Ser. Nos. 09/079,819, 09/079,723 AND 09/079,678, AND PCTAPPLICATION PCT/US98/10088, PUBLISHED AS WO 98/51325) on the transportof the dipeptide ¹⁴C-gly-sar and of the reporter molecule ³H-fMLP acrossCaco-2 monolayers was determined. The Caco-2 monolayers were grown onTranswell-Snapwells. Cell viability was determined by measuring TEER ofthe Caco-2 monolayers during each experiment. No significant drop inTEER was measured. Cell permeability was determined by measuringmannitol flux across the Caco-2 monolayers during each experiment. Noincrease in mannitol flux was measured in the presence of the MTLPZElan094.

[0251] The flux of the dipeptide ¹⁴C-gly-sar and of the reportermolecule ³H-fMLP across the Caco-2 monolayers in the absence and in thepresence of the MTLPs ZElan094, Elan178 and ZElan187 (SEQ ID NOS: 2, 7and 16) and of the targeting peptide ZElan022 (U.S. patent applicationSer Nos. 09/079,819, 09/079,723 AND 09/079,678), AND PCT APPLICATIONPCT/US98/10088, PUBLISHED AS WO 98/51325 was measured over 2 h, andreduction in the permeability coefficient was; determined in thepresence of cold substrates.

[0252] As shown in Table 6, the MTLPs ZElan 094,178 and 187 inhibitedtransport of the reporter molecule ³H-fMLP (FIG. 4), but did not inhibittransport of the dipeptide ¹⁴C-gly-sar. The targeting peptide ZElan 022inhibited transport of the reporter molecule ³-fMLP (FIG. 4). Theability of the MTLPs ZElan094, 178 and 187 to compete for the transportof fMLP across polarised Caco-2 cells indicates that this noveltransport assay can be used to screen derivatives, fragments, motifs,analogs and peptidomimetics of ZElan094 and small organic moleculesfunctionally similar to ZElan094 to identify those having improvedtransport characteristics. TABLE 6 Transport studies % inhibition³H-fMLP % inhibition ZElan N0: SEQ ID NO: transport ¹⁴C-gly-sartransport 094 2 77.2 NS 178 7 71.5 NS 187 16 84.5 NS 022 U.S. PATENT00.0 APPLICATIONS NOS. 09/079,819, 09/079,723 AND 09/079,678, , AND PCTAPPLICATION PCT/US98/10088, PUBLISHED AS WO 98/51325

[0253] NS: no significant difference between experimental (+MTLP) andcontrol cells (−MTLP) in the transport of radiolabeled drug.

[0254] Moreover, that the MTLPs inhibited transport of the reportermolecule ³H-fMLP, but did not inhibit transport of the dipeptide¹⁴C-gly-sar suggest that their effect on fMLP transport is not due to ageneralized perturbation of the membranes in polarized epithelial cells.Further, as fMLP is known to play a role in inflammation in the GIT,MTLPs, which decrease transport of fMLP across Caco-2 monolayers, mayhave a therapeutic role in preventing local inflammation by decreasingthe chemoattractant effect of fMLP in the GIT.

EXAMPLE 12 Effect of Increasing Concentrations of an MTLP on theTransport of ³H-fMLP Across c Cell Layer

[0255] Caco-2 monolayers were grown and tested for viability as inExample 11. Transport of ³H-fMLP across Caco-2 monolyers was measured inthe presence from 0 to 200 μg/ml of the MTLP ZElan094. As shown in FIG.5, the MTLP ZElan094 inhibited ³H-fMLP transport even at the lowestconcentration (13 mg/ml or 7.1 ml) tested. This indicates that the MTLPZElan094 is a potent inhibitor of fMLP transport across an epithelialcell layer.

EXAMPLE 13 Stability of MTLPs in Simulated Intestinal Fluid

[0256] MTLPs ZElan094 and ZElan207 (SEQ. ID NO: 2 AND 102)were dissolvedin water and mixed with simulated intestinal fluid pH 6.8 containingporcine derived pancreatin (SIF+Pancreatin) at 37° C. The mixtures wereincubated for up to 60 minutes at 37° C., with aliquots taken atdesignated time points. The reaction was quenched with quenchingsolution after the relevant time points in order to halt the reactionbetween the SIF and ligand.

[0257] The composition of Simulated Intestinal Fluid was as follows:

[0258] Amylase 25 USP Units

[0259] Lipase 2.0 USP units

[0260] Protease 25 USP units

[0261] (Sigma P8096)

[0262] Approximately 1 mg of ligand was dissolved in 1 ml of water. Thisstandard stock solution of ligand was used to prepare the solutionscontaining Ligand+SIF+Pancreatin. 50 μL of ligand solution was mixedwith volumes of SIF+Pancreatin in separate eppendorfs, as follows: TABLE7 Volume of Ligand Solution Volume Temperature (μL) of SIF (μL) (° C.)Time (minutes) 50 100 37  5 50 100 37 10 50 100 37 30 50 100 37 60 50200 37  5 50 200 37 10 50 200 37 30 50 200 37 60

[0263] Two different volumes of SIF were utilised in order to monitor ifincreasing the SIF:ligand ratio had an effect on the extent and rate ofdegradation. At the appropriate time point the reaction between theligand and SIF was stopped by pipetting 100 μL of the mixture into 500μL of quenching solution (Acetonitrile: Water 30:70). 20 μL of themixture was injected onto the HPLC system.

[0264] HPLC Experimental Column: Jupiter C₁₈ RP, 5 μm, 300Å, 250 × 4.6mm, TCD #188 Mobile phase: A: 10% acetonitrile in 0.1% trifluoroaceticacid in water B: 0.1% trifluoroacetic acid in acetonitrile Flow rate: 1.0 ml/min Temperature: ambient Injection  20 μl volume: Detector λ:220 nm Run time:  38 minutes

[0265] Control samples of ligand in water and in quenching solution wereprepared to check for recovery of ligand in the absence ofSIF+Pancreatin.

[0266] No recovery for ligand ZELAN094 (SEQ ID NO 2) was obtained at anyof the time points. Degradation products were seen in the chromatogram.

[0267] No result table is presented for ZELAN094 as no recovery wasobtained for an,y of the time points. This is demonstrated by thedisappearance of the peptide peak. This ligand is therefore degradedalmost immediately on contact with SIF medium. Degradation productsappear at retention times 12.258, 13.55 and 14.067 minutes.

[0268] The controls demonstrated that the peptide is not degraded at 37°C. over timea when SIF+Pancreatin are not present. Also, the quenchingsolution does not affect the recovery. It should be remembered thatwhile the HPLC method used above has not been optimised as astability-indicating assay, the disappearance of the ligand peaks andappearance of new component peaks were clearly visible.

[0269] The recovery of ZELAN207 (SEQ ID NO 102) from SIF solutions istabulated below. TABLE 8 Stability of peptide ZELAN 207 (Lot# 11243) inpresence of SIF(USP) pH 6.8 at 37 C Actual Theoretical Time Std Std ConcDrug point Std area precision Conc Sample Drug Conc % Sample (min)(mean) (% CV) (ug/ml) Area (ug/ml) (ug/ml) Recovery Peptide + 100 uL SIF 5 2012290 0.9 60.63 1727236 52.04 53.9 96.6 10 2012290 0.9 60.631742290 52.49 53.9 97.4 30 2012290 0.9 60.63 1571456 47.35 53.9 87.8 602012290 0.9 60.63 1747950 52.67 53.9 97.7 Peptide + 200 uL SIF  52013467 1.0 60.63 1062099 31.98 32.3 99.0 10 2013467 1.0 60.63 103259731.09 32.3 96.3 30 2013467 1.0 60.63  995036 29.96 32.3 92.8 60 20134671.0 60.63 1035460 31.18 32.3 96.5 Pep + H2O(100 ul) + Q* 60 2016394 1.160.63 1810419 54.44 53.9 101.0 Pep H2O(100 ul) + H2O 60 2016394 1.160.63 1806474 54.32 53.9 100.8

[0270] Incubation with SIF for up to 1 hour allowed recovery of >90% ofthe parent peptide indicating that D-amino acid substitutionsubstantially increased the stability profile for the peptide.

EXAMPLE 14 Evaluation of MTLPs for Delivery of a Model Opioid Peptide InVitro (Caco-2) and In Vivo (Intraduodenal; Conscious Rat Model)

[0271] A. Opioid Peptide Stability in SIF

[0272] A model D-form opioid peptide (H-ffir-NH2; kappa receptorspecific; molecular weight 581 Da) was evaluated for stability in SIF.

[0273] Pancreatin (Fisher Scientific) was dissolved at 1 mg/ml in1×phosphate buffer solution. The pH of the solution was adjusted to 7.5with 0.01 M NaOH and it was heated in a waterbath to 37° C. Two drypeptide samples were weighed out. One sample was dissolved in phosphatebuffer solution at 1.0 mg/ml as a control. The second sample wasdissolved in SIF at 1.0 mg/ml for stability analysis.

[0274] HPLC Analysis Conditions

[0275] RP-HPLC analysis with C-18 short column (Betasil column, 5 μm(50×3 mm) PN: 055-701-3) with the following gradient: Time (min)Solvents Mixture 0 95% water-5% acetonitrile (0.05% TFA) 0 95% water-5%acetonitrile (0.05% TFA) 0 5% water-95% acetonitrile (0.05% TFA) linearsolvent gradient 8 5% water-95% acetonitrile (0.05% TFA)

[0276] Diode array detector on the system, 214 nm used for analysis

[0277] Peptide control initially injected

[0278] Solution in SIF injected at 0, 1, 3, and 24 hours

[0279] LCMS Analysis

[0280] Analyzed control and 24 hour sample by LCMS.

[0281] The kappa peptide, H-ffir-NH2, was very stable over the 24 hourperiod (see Table 9). Mass spectrometry confirmed this result with onlyone compound detectable initially and after 24 hours. The kappa peptidewas selected as a suitable model drug for further evaluation intargeting studies. TABLE 9 Control Initial 1 hr 24 hr H-ffgr-NH2 RT AreaRT Area RT Area RT Area 0.467 347767 0.467  336841 0.467  360462 0.467 330859 2.133  249457 2.117  285901 2.333 2.317 11107788 2.317 116911062.317 11215885 Sequence H- f f i r —NH2 % Compound 1 hr 105.25% 24 hr100.97%

[0282] B. Synthesis and Evaluation of Kappa Peptide Conjugates In Vitro

[0283] Conjugates of the kappa peptide, H-ffir-NH2, and various MTLPs(ELAN094, ELAN207, ELAN208 & ELAN 178) were synthesised in variousformats to determine optimal conjugation strategies for subsequentsynthesis of batches for in vivo evaluation. Structural formatsincluded:

[0284] i) C-terminal or N-terminal opioid peptide,

[0285] ii) Conjugated with I without a lysine linker,

[0286] iii) Unlabelled I biotin labelled.

[0287] The integrity of the opioid peptide was evaluated postconjugation to assess suitability for inclusion in further studies.Opioid activity was assessed in vitro using a competition assay(competition for binding of a radiolabelled kappa peptide to rat brainhomogenates). Results are expressed as IC50 values i.e. theconcentration of conjugate which inhibits binding of the radiolabelledligand by 50%. TABLE 10 Day 1 Day 5 Day 82 Cmpnd # M.W. IC50 nM IC50 nMIC50 nM IC50 nM IC50 nM IC50 nM P10- 110H-ffirk(kkaaavllpvllaap-NH-e)-NH2 2166 52.1 81.8 181.8 25.9 17.1 19704.9P10- 114 H-kkaaavllpvllaapk(ffir-NH-e)-NH2 2166 3.7 19.9 1.0 0.8 0.8P10- 118 H-ffirkkaaavllvllaap-NH2 2038 113.1 63.0 302.5 177.1 38.8H-ffir-NH2 14.0 3.3 4 1.7 3.9

[0288] In Table 10, 3 Elan207 (SEQ ID NO 102) conjugates are compared tothe H-ffir-NH2 peptide control (unconjugated). The three conjugatesare 1) H-ffir-NH2 conjugated to N-terminal of 207 through a lysinelinker (P10-110), 2) H-ffir-NH2 conjugated to C-terminal of 207 througha lysine linker (P10-114) and 3) H-ffir-NH2 conjugated to N-terminal of207 directly i.e. no linker (P10-118). Assays were performed on 3separate occasions (duplicates on day 5 and triplicates on day 82).Opioid activity is significantly reduced by conjugation as describedin 1) and 3). Using 2) i.e. C-terminus and lysine linker the opioidactivity is retained, or even enhanced suggesting the need for thelinker amino acid and/or conformation induced in this format. The assayis cell based and subject to some variation—however the trend remainsconsistent throughout.

[0289] IC50 values indicated that optimal opioid activity was retainedpost conjugation of the opioid peptide to the C-terminus of the ELAN207MTLP through a lysine linker. Note that the opioid activity may havebeen enhanced by addition of ELAN207.

[0290] In Table 11, ELAN094, 178, 207 & 208 (SEQ ID NOs 2, 7, 102 & 202)biotin labelled conjugates are compared to the H-ffir-NH2 peptidecontrol (unconjugated). TABLE 11 ELAN No. MW IC50 nM IC50 nM P37-114H-K(biotin-LC)KKAAAVLLPVLLAAPK(ffir-NH-e)-NH2  94 2633 0.4 7.9 P37-116H-K(biotin-LC)KKCAAVLLPVLLACK(ffir-NH-e)-NH2 178 2598 0.2 11.7  P34-154H-K(biotin-LC)kkaaavllpvllaapk(ffir-NH-e)-NH2 207 2633 1.1 12.4  P37-115H-K(biotin-LC)paallvpllvaaakkK(ffir-NH-e)-NH2 208 2633 9.2 9.6H-ffir-NH2 1.7 3.9

[0291] IC50 values indicated that:

[0292] i) the ELAN207 opioid peptide conjugate (P34-154) retainedactivity post biotin labelling

[0293] ii) ELAN094 (P37-114), 178 (P37-116) & 208 (P37-115) opioidpeptide conjugates, with C-terminal opioid peptide and lysine linker,exhibited activity equivalent to that observed with ELAN207 (P34-154).

[0294] Peptide conjugates P37-114 (ELAN094) and P34-154 (ELAN207) weresynthesised in a tritium labelled format for in vivo studies.

EXAMPLE 15

[0295] C. Synthesis and Evaluation of Tritiated Kappa Peptide ConjugatesIn Vitro

[0296] Conjugates of the kappa peptide, H-ffir-NH2, and MTLPs ELAN094and ELAN207 (SEQ ID NOs 2, 102), were synthesised using standard peptidesynthesis protocols. Two phenylalanine residues on the kappa peptidewere labelled by tritium exchange and radio-peptide purity was assessedby RP-HPLC. Peptide purity of >95% and specific activities of 42-54Ci/mmol were achieved.

[0297] The tritiated opioid peptide conjugates were evaluated forpermeability through differentiated Caco-2 cell monolayers i.e. toassess the integrity of the membrane translocating peptide moiety Boththe kappa peptide conjugates and the kappa peptide control exhibitedpermeability coefficients in the 10⁻⁶ cm/sec range, indicating that theywould be suitable candidates for oral bioavailability evaluation invivo. No significant increase in permeability was detectable when theZELAN094 or ZELAN207 peptide conjugates were compared to the kappapeptide control (n=5). Highest transport values were obtained in thefirst 30 min followed by a 2-4 fold decrease at 60 min. The significanceof these findings is unclear. The data may be indicative of dualbinding/uptake events occurring through i) a kappa receptor specificmechanism and ii) direct membrane interaction through lipid interactionof the membrane permeable peptide.

[0298]FIG. 6 shows results on the transport of tritiated kappa peptideconjugates and kappa peptide control across differentiated Caco-2 cellmonolayers.

[0299] D. Evaluation of Tritiated Kappa Peptide Conjugates In Vivo

[0300] The possible enhancing effects of ELAN094 and ELAN207 MTLPs (SEQID NOs 2, 102) on intestinal absorption of the kappa peptide wereexamined in a conscious rat model.

[0301] Experimental:

[0302] The study was non-randomised, parallel group design. Wistar ratswithin the 250-350 g weight range were used. All animals were fasted fora period of 16 h prior to study initiation. Water was available at alltimes.

[0303] Treatment Regimen:

[0304] Group 1 (n=6)

[0305] Intravenous injection of 10 μCi of ³H-kappa peptide-Kaffiralin-1(plain kappa peptide) (tail vein injection).

[0306] Group 3 (n=6)

[0307] Intraduodenal instillation of 100 μCi of ³H-kappa peptide-Elan094(Analysed). The Elan094 ligand contains a membrane translocatingsequence.

[0308] Group 4 (n=6)

[0309] Intraduodenal instillation of 100 μCi of ³H-kappa peptide-Elan207(Analysed). The Elan207 ligand contains a membrane translocatingsequence.

[0310] Group 10 (n=6)

[0311] Intraduodenal instillation of 100 μCi of ³H-kappapeptide-Kaffiralin-1.

[0312]³H Bio-analysis: Plasma Samples

[0313] The plasma (100-250 μl) was made up to 1 ml with BTS-450 (anorganic tissue solubiliser) and incubated for 1 hour. Ten ml ofScintillation fluid was added and the radioactivity was measured.

[0314] Results:

[0315] The study was a non-randomised, parallel group biostudy designedto evaluates the systemic bioavailability of tritium labelled kappapeptide conjugates after intra-duodenal instillation in the consciousrat model. Each rat only received one treatment and the plasma sampleswere analysed for tritium content. The calculation of AUClast, Amax,tmax, Volume of Distribution (V d) and Clearance was based on base linecorrected data. The absolute bioavailability was calculated usingpotency corrected data.

[0316] Stock solutions of the tritiated ligand were administered to therats on different dates. These stock solutions were analysed in order tocorrect for potency. Data analysed on one particular day suggested adiscrepancy in the bioavailability for Group 4 and a repeat of Group 4.It was decided to re-analyse the stock solution administered to Group 4(repeat). The potency correction factor for all treatments, includingthe re-analysis of Group 4(repeat), are summarised below:

[0317] Potency Correction Analysis: TABLE 12 Theoritical Actual % ofPotency Day Value Group Value theo Corr. received uCi/ml No Liganddpm/100 ul dpm/ml uCi/ml dose Factor  1 303 10 Kappa-peptide 74811764748117640 336.990 111.22 0.899  6 303 10 Kappa-peptide 47279266472792660 212.970 70.29 1.423 13 303 4 Elan 207 83174222 831742220374.659 123.65 0.809 21 303 4 Elan 207 83186452 831864520 374.714 123.670.809 27  50 1 Kappa-peptide 12839706 128397060 57.837 115.67 0.865 48303 3 Elan094 32985636 329856360 148.584 49.04 2.039 62 303 4(Repeat)Elan207 44012732 440127320 198.256 65.43 1.528 113  303 4(Repeat)Elan207 88311270 883112700 397.799 131.29 0.762

[0318] Day numbers are unrelated to those in Table 10.

[0319] The absolute bioavailability and mean pharmacokinetic parametersare summarized below.

[0320] Absolute ³H Bioavailability (%)

[0321] The absolute ³H bioavailability for the treatments in rank orderwere the following: for Treatment 3—100 μCi ³H-Kappa peptide-Elan094(ID) was 46.48±6.24% (CV 13.4%), for Treatment 4—100 μCi ³H-Kappapeptide Elan207 (ID) (Repeat) was 16.60±2.50% (15.1%), for Treatment4—100 μCi ³H-Kappa peptide Elan207 (ID) (original) was 11.52±0.96% (CV8.3%), and in the absence of delivery enhancer ligand Treatment 10—100μCi ³H-Kappa peptide Kaffiralin-1 (ID) was 7.94±1.92% (CV 24.1%). Thisrepresents approximately a 6 fold increase in delivery with Treatment3—100 μCi ³H-Kappa peptide-Elan094 (ID), approx. 2 fold increase withTreatment 4—100 μCi ³H-Kappa peptide Elan207 (I D) (Repeat). A similardelivery was observed with Treatment 4—100 μCi ³H-Kappa peptide Elan207(ID) (original) as compared to the absolute ³H bioavailability ofKappa-peptide in the absence of enhancer ligand Treatment 10—100 μCi³H-Kappa peptide Kaffiralin-1 (ID).

[0322]³H AUClast (dpm/ml.h)

[0323] The AUClast for the ID treatments in rank order were as follows:for Treatment 3—100 μCi ³H-Kappa peptide-Elan094 (ID) was260642.53±35010.58 dpm/ml.h (CV 13.4%), for Treatment 4—100 μCi 3H-Kappapeptide Elan207 (ID) (Repeat) was 249021.32±37503.78 dpm/ml.h (CV15.1%), for Treatment 4—100 μCi ³H-Kappa peptide-Elan207 (ID) (original)was 162743.70±13549.10 dpm/ml.h (CV 8.3%), for Treatment 1—10 μCi ³HKappa Peptide-Kaffiralin-1 (IV) was 132184.24±13288.32 dpm/ml.h (CV10.1%), while in the absence of ligand Treatment 10—100 μCi ³H-Kappapeptide Kaffiralin-1 (ID) was 73098.43±9493.88 dpm/ml.h (CV % 13.0). ³HAmax (dpm/ml)

[0324] The maximum radioactivity following administration of the IDtreatments in order of magnitude were as follows: for Treatment 1—10 μCi³H Kappa Peptide-Kaffiralin-1 (IV) was 903603.03±186855.31 dpm/ml (CV20.7%), for Treatment 4—100 μCi ³H-Kappa peptide Elan207 (ID) (Repeat)was 201464.40±44854.19 dpm/ml (CV 22.3%), for Treatment 3—100 μCi³H-Kappa peptide-Elan094 (ID) was 158512.67±18907.79 dpm/ml (CV % 11.9),for Treatment 4—100 μCi ³H-Kappa peptide-Elan207 (ID) (original) was100041.17±6274.57 dpm/ml (CV % 6.3), for Kappa-peptide alone Treatment10—100 μCi ³H-Kappa peptide Kaffiralin-1 (ID) was 70925.33±23631.28dpm/ml (CV % 33.3).

[0325] H tmax (h)

[0326] The time to reach maximum radioactivity was 0.08±0.00 h for allID treatments.

[0327] Volume of Distribution (ml)

[0328] The observed volume of distribution for Trt 1 10 μCi 3H-Kappapeptide-Kaffiralin-1 (IV) was 963.87±255.65 ml (CV 26.5%).

[0329] Clearance (mi/h)

[0330] The observed clearance for Trt 1 10 μCi 3H-Kappapeptide-Kaffiralin-1 (IV) was 142.85±17.24 ml/h (CV 12.1%).

[0331] The Elan207 MTLP (SEQ ID NO 102) showed absolute ³Hbioavailability in excess of 1.5 fold increase respectively incomparison to administration of the kappa peptide control. Followingre-analysis of the stocks for Group 4 (repeat), the recalculatedabsolute bioavailability was comparable to that observed for Group 4(original). The correlation of this repeat data, together with theobserved stability of the ELAN207 and kappa peptides in simulatedintestinal fluid, would suggest that the observed radioactivity isassociated with presence of the intact radio-peptide conjugate in theplasma. The radioactivity profiles will be correlated with LCMS analysisof plasma samples exhibiting high tritium counts for verification.

[0332] The Elan094 MTLP (SEQ ID NO 2) showed absolute ³H bioavailabilityin excess of approximately 5.8 fold compared to administration of thekappa peptide control. Note that the inherent instability of the ELAN094peptide in simulated intestinal fluid would suggest that theradioactivity profile should be interpreted with caution in thisinstance. It is possible that the parent ELAN094 kappa peptide conjugatehas deteriorated in vivo and thus, the observed radioactivity may not beassociated with presence of the intact radio-peptide conjugate in theplasma.

[0333] A rapid delivery of ³H kappa-peptide was detected in alltreatments with observed tmax of 0.08 h for all treatments. Thissuggests that the kappa peptide rapidly crossed the gastrintestinalbarriers into the systemic circulation. It should be noted that as theabsorption was very rapid, the AUC might be underestimated due to thefact that the maximum radioactivity was measured in the first samplingpoint (5 minutes after dosing).

[0334] 5³H Bio-analysis: Tissue Samples

[0335] The tissue was weighed and a representative sample, (ca 0.1 g),was removed to a scintillation vial. The tissue was solublized with 1 mlof BTS-450 (an organic tissue solubliser). Ten ml of scintillation fluidwas added and the radioactivity was measured. The radioactivity wasexpressed as dpm/initial weight of tissue.

[0336] Results:

[0337] The results are summarized in Table 14. TABLE 14 Summary Table %of administered dose (Mean ± SD − CV %) (Potency corrected data) Group 1Group 4 Group 10 10 μCi 3H- Group 3 100 μCi 3H- 100 μCi 3H- Kappa 100μCi 3H- Kappa Kappa peptide- Kappa peptide-Elan peptide- Kaffiralin-1peptide-Elan094 207 Kaffiralin-1 % of Admin. (IV) (IDV) (ID) (ID) Dose n= 6 n = 6 n = 6 n = 6 *Total 34.43 ± 13.36 46.00 ± 19.91 40.91 ± 23.3261.79 ± 16.93 Recovery (%) (CV %) 38.8 43.3 57.0 27.4 Catheter — 0.20 ±0.10 0.14 ± 0.09 0.06 ± 0.05 (%) (CV %) — 49.3 63.4 88.8 Plasma 0.16 ±0.03 0.42 ± 0.08 0.10 ± 0.01 0.06 ± 0.01 (%) (CV %) 18.9 17.7  9.2 11.6Liver 6.66 ± 1.77 0.37 ± 0.06 0.47 ± 0.20 0.63 ± 0.37 (%) (CV %) 26.617.3 41.9 57.9 Kidney 0.51 ± 0.08 0.12 ± 0.07 0.17 ± 0.07 0.16 ± 0.17(%) (CV %) 16.1 54.1 40.6 109.6  GI Tissue 1.62 ± 0.41 11.61 ± 11.274.08 ± 2.47 7.72 ± 4.98 (%) (CV %) 25.4 97.1 60.4 64.5 GI contents 23.50± 11.04 23.57 ± 21.57 26.34 ± 17.00 37.31 ± 15.75 (%) (CV %) 47.0 91.5064.5 42.2 GI Washing 1.98 ± 1.01 9.70 ± 5.24  9.60 ± 16.65 15.85 ± 5.00 (%) (CV %) 51.1 54.1 173.4  31.5 Total GI 27.10 ± 11.93 44.89 ± 19.9440.03 ± 23.23 60.87 ± 16.74 (% recovery from GI tissue, GI contents & GIwash) (CV %) 44.0 44.4 58.0 27.5

[0338] The tissue distribution following IV administration in rank orderwere as follows: GI contents>Liver>GI Washing>GI Tissue>Kidney>Plasma.Assuming the ³H label remained attached to the Kappa-peptide, this datasuggests that following IV administration the administered ³HKappa-peptide-Kaffiralin-1 had widely distributed throughout the rat andwas concentrated mainly in the GI, particularly in GI contents. Theconcentration in GI contents may possibly be due to biliary excretion.However only 34.43±13.36% (CV 38.8%) of administered dose had beenrecovered at t=6 h suggesting some Kappa-peptide had been excreted orhad possibly distributed to other sites.

[0339] % Recovered in Catheter

[0340] The % of administered dose recovered in the catheter, for thevarious treatments, ranged from 0.06-0.39%. This data suggests that anegligible amount of the administered dose is lost in the catheter.

[0341] % Recovered in Plasma

[0342] The % of administered dose recovered in plasma was calculated att=6 h. The % of administered dose ranged from 0.06-0.42% for the varioustreatments. The rank order for the various treatments were as follows:for Treatment 3—100 μCi ³H-Kappa peptide-Elan094 (ID) was 0.42±0.08%(17.7%), for Treatment 1—10□Ci ³H Kappa peptide-Kaffiralin-1 (IV) was0.16±0.03% (CV 18.9%), for Treatment 4—100 μCi ³H-Kappa peptide Elan207(ID) (original) was 0.10±0.01% (CV 9.2%), and in the absence of deliveryenhancer ligand Treatment 10—100 μCi ³H-Kappa peptide Kaffiralin-1 (ID)the % of administered dose was 0.06±0.01% (CV 11.6%). This rank ordercorrelates identically with that observed for absolute bioavailabilityreported in the data pack for Biostudy 1000003 for the varioustreatments.

[0343] % Recovered in Liver

[0344] The % of administered dose recovered in the liver ranged from0.26-6.66%. Following IV administration, Treatment 1—10 μCi ³H Kappapeptide-Kaffiralin-1 (IV), the % recovery was 6.66±1.77% (CV 26.6%).This recovery far exceeded the amount recovered in liver tissue for anyother treatments.

[0345] % Recovered in Kidney Tissue

[0346] The % of administered dose recovered in kidney ranged from0.12-0.51% for the various treatments. This data suggests a negligibleamount of administered dose of ³H Kappa-peptide had accumulated inkidney tissue at t=6 h.

[0347] % Recovered in GI Tract

[0348] The % of administered dose recovered in the GI tract ranged from27.10-89.26%. The GI tract can be further sub divided into GI tissue, GIcontents and GI Washing, the range for each was 1.62-11.97%,23.50-65.68% and 1.98-25.84% respectively. The data presented as % ofadministered dose recovered in the GI Tract represents the summation of6 segments of GI tissue. Within the GI tract the highest levels ofradioactivity were associated with the latter segments (refer to rawdata table). The recovered radioactivity associated with GI segment 6and its corresponding GI contents were higher than that observed forother segments.

[0349] In summary, the greatest % of administered dose of ³HKappa-peptide-ligand conjugates were recovered primarily in the GI tractand more specifically in GI contents. Assuming that the ³H labelremained attached to the Kappa-peptide, not all the administered dosewas recovered suggesting that the Kappa-peptide-ligand-conjugates mayhave been excreted or had distributed to other sites within the rat. Itmust be noted that other potential sites for distribution, such as CNStissue, weren't analysed in this study. These measurements are of ³HKappa-peptide only and not of intact conjugate.

[0350] The present invention is not to be limited in scope by thesepcific embodiment, described herein. Various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

We claim:
 1. A composition comprising a translocating peptide, saidtranslocating peptide selected from the group consisting of a transportpeptide, an extended peptide comprising said transport peptide, and atransport-active fragment of at least 4 amino acids of said transportpeptide, wherein said transport peptide is selected from the groupconsisting of an L-peptide, a d-peptide, and a retroinverted peptide,and wherein said L-peptide has an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 2-13, and 15-24, wherein said d-peptidehas an amino acid sequence selected from the group consisting of SEQ IDNOS. 102-124 corresponding to the d-forms of L-peptides of SEQ ID NOS.2-24, and wherein the retroinverted peptide has an amino acid sequenceselected from the group consisting of a peptide of SEQ ID NOS. 202-224,corresponding to retroinverted forms of L-peptides of SEQ ID NOS: 2-24.2. A composition of claim 1, wherein said L-peptide has an amino acidsequence selected from the group consisting of SEQ ID NOS: 2-4, 16, 23and
 24. wherein said d-peptide has an amino acid sequence selected fromthe group consisting of SEQ ID NOS. 102-104, 116, 123 and 124corresponding to the d-forms of L-peptides of SEQ ID NOS. 2-4,16, 23 and24, wherein the retroinverted peptide has an amino acid sequenceselected from the group consisting of a peptide of SEQ ID NOS. 202-204,216, 223 and 224, corresponding to retroinverted forms of an L-peptidesof SEQ ID NOS: 2-4, 16, 23 and
 24. 3. A composition of claim 1, whereinsaid transport peptide is partially or completely cyclic.
 4. Acomposition of claim 3 wherein any fragment of said transport peptide isalso partially or completely cyclic.
 5. A composition of claim 4 wherein the wherein said L-peptide has an amino acid sequence selected fromthe group consisting of SEQ ID NOS: 5-13; wherein said d-peptide has anamino acid sequence selected from the group consisting of SEQ ID NOS.105-113 corresponding to the d-forms of L-peptides of SEQ ID NOS. 5-13,wherein the retroinverted peptide has an amino acid sequence selectedfrom the group consisting of a peptide of SEQ ID NOS. 205-213,corresponding to retroinverted forms of L-peptides of SEQ ID NOS: 5-13.6. A composition of claim 1, wherein said translocating peptide is anextended peptide of a transport peptide.
 7. A composition of claim 6wherein the extended peptide is not more than 100 amino acids in length.8. A composition of claim 7 wherein the extended peptide is not morethan 50 amino acids in length.
 9. A composition of claim 1 wherein thetranslocating peptide is a transport peptide.
 10. A composition of claim1 where in the transport-active fragment is at least 6 amino acids of atransport peptide.
 11. A composition of claim 1 where in thetransport-active fragment is at least 8 amino acids of a transportpeptide. 11A. A composition of claim 1 wherein the translocating peptideis selected from the group consisting of Elan 094, Elan178, Elan207, andElan208.
 12. A composition of claim 1 wherein the carboxyl end group ofthe translocating peptide has been modified to create an amide group.13. A composition comprising a translocating peptide, said translocatingpeptide selected from the group consisting of a transport peptide, anextended peptide comprising said transport peptide, and atransport-active fragment of at least 4 amino acids of said transportpeptide, said transport peptide being an L-peptide that has an aminoacid sequence SEQ ID NO: 14 blocked at its carboxyl end with an amidegroup and wherein any of said fragments is also blocked at its carboxylend with an amide group.
 14. The composition of claims 1 or 13, furthercomprising an active agent, wherein the translocating peptide iscomplexed to an active agent to form a translocating peptide-activeagent complex.
 15. The composition of claims 1 or 13, further comprisingan active particle, wherein the translocating peptide is complexed tothe active particle to form a translocating peptide-active particlecomplex.
 16. A method for enhancing movement of an active agent across alipid membrane, comprising using a translocating peptide-active agentcomplex, wherein the translocating peptide enhances movement of theactive agent across the lipid membrane.
 17. A method for enhancingmovement of an active particle across a lipid membrane, comprising usinga translocating peptide-active particle complex, wherein thetranslocating peptide enhances movement of the active particle acrossthe lipid membrane.
 18. A method for identifying a compound havingenhanced ability to transport an active agent across a lipid membrane,wherein the compound competes with the translocating peptide fortransport of an fMLP across a membrane selected from the groupconsisting of a cell membrane, an intracellular membrane, the apical andbasal membranes of an epithelial cell layer.
 19. The method of claim 18,wherein the epithelial cell layer is a polarized epithelial cell layer.20. A method for treating a pathological disorder in an animal,comprising orally administering to the animal in need of such treatmenta complex selected from the group consisting of a translocatingpeptide-active agent complex and a translocating peptide-active particlecomplex, wherein an amount of the active agent effective to treat thepathological disorder is moved across the gastrointestinal epithelium ofthe animal into the circulation.
 21. A chimeric polypeptide comprising(A) a translocating peptide of claims 1 or 13, (B) a translocatablepeptide, and (C) an amino acid linker sequence that directly links thetranslocating peptide to the translocatable peptide, wherein saidtranslocatable peptide is between 3 and 200 amino acids, and whereinsaid amino acid linker sequence is between 1 and 20 amino acids.
 22. Achimeric peptide of claim 21 wherein said translocatable peptide isbetween 3 and 30 amino acids.
 23. A chimeric peptide of claim 21 whereinthe translocatable peptide is an opioid peptide.
 24. A chimeric peptideof claim 21 wherein said linker sequence is not more than 7 amino acids.25. A chimeric peptide of claim 24 wherein said linker sequence is notmore than 3 amino acids.
 26. A chimeric peptide of claim 25 wherein saidlinker sequence is 1 amino acids.
 27. A chimeric peptide of claim 26wherein said at least 50% of the amino acids in the linker sequence arelysines.
 28. A chimeric peptide of claim 26 wherein said at least 80% ofthe amino acids in the linker sequence are lysines.
 29. A chimericpeptide of claim 26 wherein all of the amino acids in the linkersequence are lysines.
 30. A method of delivering a chimeric peptide tothe blood, said method comprising orally administering a chimericpeptide of claim
 21. 31. A nucleic acid molecule coding for atranslocating peptide of claim
 1. 32. A nucleic acid molecule of claim31 wherein the translocating peptide is an L-form peptide.
 33. A nucleicacid molecule coding for a chimeric protein of claim
 21. 34. A nucleicacid molecule of claim 33 wherein the chimerica peptide is an L-formpeptide.
 35. A chimeric constructs comprising (A) a translocatingpeptide of claims 1 or 13, (B) a translocatable peptide, and (C) annon-amino acid linker that directly links the translocating peptide tothe translocatable peptide, wherein said translocatable peptide isbetween 3 and 200 amino acids.
 36. A method of delivering a chimericconstruct to a site within a person, said method comprisingadministering a chimeric construct of claim 35, said site being selectedfrom the group consisting of a tissue, a fluid, a cell, and asub-cellular compartment.